Microspotting Device

ABSTRACT

Devices and methods are provided for spotting an array with fluid. Arrays produced by such methods are also provided. In one aspect of the invention, a spotter device for spotting a plurality of fluids into an array is described, the spotter device comprising a plurality of reservoirs provided in a first configuration, each reservoir holding its respective fluid, a print head having a plurality of positions provided in a second configuration, the second configuration being different from the first configuration, a plurality of tubes, each tube configured to provide fluid communication from a reservoir at a first end of the tube to a position in the print head at the second end of the tube, and a pump for pumping fluid through the tubes from the reservoir to the print head.

GOVERNMENT INTEREST

This invention was made with government support under Grant No.AI061611, awarded by National Institutes of Health. The U.S. Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

In the United States, Canada, and Western Europe infectious diseaseaccounts for approximately 7% of human mortality, while in developingregions infectious disease accounts for over 40% of human mortality.Infectious diseases lead to a variety of clinical manifestations. Amongcommon overt manifestations are fever, pneumonia, meningitis, diarrhea,and diarrhea containing blood. While the physical manifestations suggestsome pathogens and eliminate others as the etiological agent, a varietyof potential causative agents remain, and clear diagnosis often requiresa variety of assays be performed. Traditional microbiology techniquesfor diagnosing pathogens can take days or weeks, often delaying a propercourse of treatment.

In recent years, the polymerase chain reaction (PCR) has become a methodof choice for rapid diagnosis of infectious agents. PCR can be a rapid,sensitive, and specific tool to diagnose infectious disease. A challengeto using PCR as a primary means of diagnosis is the variety of possiblecausative organisms and the low levels of organism present in somepathological specimens. It is often impractical to run large panels ofPCR assays, one for each possible causative organism, most of which areexpected to be negative. The problem is exacerbated when pathogennucleic acid is at low concentration and requires a large volume ofsample to gather adequate reaction templates. In some cases there isinadequate sample to assay for all possible etiological agents. Asolution is to run “multiplex PCR” wherein the sample is concurrentlyassayed for multiple targets in a single reaction. While multiplex PCRhas proved to be valuable in some systems, shortcomings exist concerningrobustness of high level multiplex reactions and difficulties for clearanalysis of multiple products. To solve these problems, the assay may besubsequently divided into multiple secondary PCRs. Nesting secondaryreactions within the primary product increases robustness. However, thisfurther handling can be expensive and may lead to contamination or otherproblems.

The present invention addresses various issues of handling materials toperform biological analysis.

SUMMARY OF THE INVENTION

In one aspect of the invention, a spotter device for spotting aplurality of fluids into an array is described, the spotter devicecomprising a plurality of reservoirs provided in a first configuration,each reservoir holding its respective fluid, a print head having aplurality of positions provided in a second configuration, the secondconfiguration being different from the first configuration, a pluralityof tubes, each tube configured to provide fluid communication from areservoir at a first end of the tube to a position in the print head atthe second end of the tube, and a pump for pumping fluid through thetubes from the reservoir to the print head. Various features of theconfiguration of the spotter device, the pump, the print head, and othercomponents are described herein.

In another aspect of the invention a method for printing an array havinga plurality of wells in a configuration is provided. The methodcomprises simultaneously pumping fluid from a plurality of reservoirs toa plurality of positions on a print head to form a plurality of drops onthe print head having the same configuration as the array, moving thearray into contact with the drops, and simultaneously transferring eachrespective drop into its respective well. Arrays manufactured by suchmethods are also disclosed.

In yet another aspect, a system is provided for delivering one or moreliquids into a preselected array of a plurality of wells, the systemcomprising a plurality of tubes, each tube having a first end in fluidcommunication with a reservoir, and a second end terminating in anorifice; a plurality of reservoirs, the reservoirs provided in apredetermined configuration relative to one another; a print headoperable to movably hold each orifice in a predetermined position suchthat the position of each orifice corresponds to a well in thepreselected array of wells; a plurality of straws, each straw having ahollow opening connecting a first end to a second end, the first endfluidly connected to the first end of a corresponding tube and thesecond end removably in contact with a bottom portion of a correspondingreservoir; and a metering device operable to urge a preselected amountof fluid from the second end of each straw through its correspondingtube, and out its corresponding orifice.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flexible pouch according to one embodiment of thisinvention.

FIG. 2 shows an embodiment of the cell lysis zone of the flexible pouchaccording to FIG. 1.

FIG. 2 a shows an embodiment of a portion of a bladder corresponding tothe cell lysis zone shown in FIG. 2.

FIG. 2 b shows an embodiment of the cell lysis zone of the flexiblepouch according to FIG. 1 having an alternative vortexing mechanism.

FIG. 3 shows an embodiment of the nucleic acid preparation zone of theflexible pouch according to FIG. 1.

FIG. 4 shows an embodiment of the first-stage amplification zone of theflexible pouch according to FIG. 1.

FIG. 5 is similar to FIG. 1, except showing an alternative embodiment ofa pouch.

FIG. 5 a is a cross-sectional view of the fitment of the pouch of FIG.5.

FIG. 5 b is an enlargement of a portion of the pouch of FIG. 5.

FIG. 6 is a perspective view of another alternative embodiment of apouch.

FIG. 6 a is a cross-sectional view of the fitment of the pouch of FIG.6.

FIG. 7 shows illustrative bladder components for use with the pouch ofFIG. 6.

FIG. 8 is an exploded perspective view of an instrument for use with thepouch of FIG. 6, including the pouch of FIG. 6.

FIG. 9 shows a partial cross-sectional view of the instrument of FIG. 8,including the bladder components of FIG. 7, with the pouch of FIG. 6shown in shadow.

FIG. 10 shows a partial cross-sectional view of the instrument of FIG.8, including various bladders for pinch valves and the pouch of FIG. 6.

FIG. 11 shows amplification curves from second-stage amplification of asample that was lysed and amplified in a pouch of FIG. 5 (—positivecontrol;—S. cerevisaie target 1;—S. cerevisaie target 2;—S. cerevisaietarget 3;—S. pombe target 1;—S. pombe target 2;—negative controls).

FIG. 12 is similar to FIG. 6, except showing a pouch having asecond-stage high density array.

FIG. 13 shows a modification of a component of the instrument of FIG. 8.A support member has been provided with a motor configured for use withthe pouch of FIG. 12.

FIG. 14 is an exploded perspective view of the second-stage high densityarray of FIG. 12.

FIG. 15 is a bottom view of the second-stage high density array of FIG.12, shown during construction of the second-stage high density array.

FIG. 16 shows a partially constructed high-density array similar to thatshown in FIGS. 14-15, except with a different arrangement of wells.

FIG. 17 shows a perspective view of a spotter with the placement armremoved to allow detail underneath to be seen.

FIG. 18 is similar to FIG. 17, except that the placement arm is shown.

FIG. 19 is a right side view of a portion of the spotter of FIG. 18.

FIG. 20 is a cross-sectional view along line 20-20 in FIG. 17.

FIG. 21 is a cross-sectional view along line 21-21 in FIG. 17.

FIG. 22 is a bottom view of the print head of FIG. 17, showing 96sufficient drops.

FIG. 23 is similar to FIG. 22 is similar to FIG. 22, except that only 76drops are shown.

FIG. 24 is a right side view of the spotter of FIG. 18, showing detailof the placement arm.

FIG. 25 is similar to FIG. 24, except that the placement arm has movedthe array into contact with the print head.

FIG. 26 is a schematic of a spotter system.

DETAILED DESCRIPTION

The self-contained nucleic acid analysis pouches described herein may beused to assay a sample for the presence of various biologicalsubstances, illustratively antigens and nucleic acid sequences,illustratively in a single closed system. In one embodiment, the pouchis used to assay for multiple pathogens. Illustratively, various stepsmay be performed in the optionally disposable pouch, including nucleicacid preparation, primary large volume multiplex PCR, dilution ofprimary amplification product, and secondary PCR, culminating withreal-time detection and/or post-amplification analysis such asmelting-curve analysis. It is understood, however, that pathogendetection is one exemplary use and the pouches may be used for othernucleic acid analysis or detection of other substances, including butnot limited to peptides, toxins, and small molecules. Further, it isunderstood that while the various steps may be performed in pouches ofthe present invention, one or more of the steps may be omitted forcertain uses, and the pouch configuration may be altered accordingly.

While PCR is the amplification method used in the examples herein, it isunderstood that any amplification method that uses a primer may besuitable. Such suitable procedures include polymerase chain reaction(PCR); strand displacement amplification (SDA); nucleic acidsequence-based amplification (NASBA); cascade rolling circleamplification (CRCA), loop-mediated isothermal amplification of DNA(LAMP); isothermal and chimeric primer-initiated amplification ofnucleic acids (ICAN); target based-helicase dependant amplification(HDA); transcription-mediated amplification (TMA), and the like.Therefore, when the term PCR is used, it should be understood to includeother alternative amplification methods. It is understood that protocolsmay need to be adjusted accordingly.

FIG. 1 shows an illustrative self-contained nucleic acid analysis pouch10. Pouch 10 has a cell lysis zone 20, a nucleic acid preparation zone40, a first-stage amplification zone 60, and a second-stageamplification zone 80. A sample containing nucleic acid is introducedinto the pouch 10 via sample injection port 12. Pouch 10 comprises avariety of channels and blisters of various sizes and is arranged suchthat the sample flows through the system. The sample passes through thevarious zones and is processed accordingly.

Sample processing occurs in various blisters located within pouch 10.Various channels are provided to move the sample within and betweenprocessing zones, while other channels are provided to deliver fluidsand reagents to the sample or to remove such fluids and reagents fromthe sample. Liquid within pouch 10 illustratively is moved betweenblisters by pressure, illustratively pneumatic pressure, as describedbelow, although other methods of moving material within the pouch arecontemplated.

While other containers may be used, illustratively, pouch 10 is formedof two layers of a flexible plastic film or other flexible material suchas polyester, polyethylene terephthalate (PET), polycarbonate,polypropylene, polymethylmethacrylate, and mixtures thereof that can bemade by any process known in the art, including extrusion, plasmadeposition, and lamination. Metal foils or plastics with aluminumlamination also may be used. Other barrier materials are known in theart that can be sealed together to form the blisters and channels. Ifplastic film is used, the layers may be bonded together, illustrativelyby heat sealing. Illustratively, the material has low nucleic acidbinding capacity.

For embodiments employing fluorescent monitoring, plastic films that areadequately low in absorbance and auto-fluorescence at the operativewavelengths are preferred. Such material could be identified by tryingdifferent plastics, different plasticizers, and composite ratios, aswell as different thicknesses of the film. For plastics with aluminum orother foil lamination, the portion of the pouch that is to be read by afluorescence detection device can be left without the foil. For example,if fluorescence is monitored in the blisters 82 of the second stageamplification zone 80 of pouch 10, then one or both layers at blisters82 would be left without the foil. In the example of PCR, film laminatescomposed of polyester (Mylar, Dupont, Wilmington Del.) of about 0.0048inch (0.1219 mm) thick and polypropylene films of 0.001-0.003 inch(0.025-0.076 mm) thick perform well. Illustratively, pouch 10 is made ofa clear material capable of transmitting approximately 80%-90% ofincident light.

In the illustrative embodiment, the materials are moved between blistersby the application of pressure, illustratively pneumatic pressure, uponthe blisters and channels. Accordingly, in embodiments employingpneumatic pressure, the pouch material illustratively is flexible enoughto allow the pneumatic pressure to have the desired effect. The term“flexible” is herein used to describe a physical characteristic of thematerial of pouch. The term “flexible” is herein defined as readilydeformable by the levels of pneumatic pressure used herein withoutcracking, breaking, crazing, or the like. For example, thin plasticsheets, such as Saran™ wrap and Ziploc® bags, as well as thin metalfoil, such as aluminum foil, are flexible. However, only certain regionsof the blisters and channels need be flexible, even in embodimentsemploying pneumatic pressure. Further, only one side of the blisters andchannels need to be flexible, as long as the blisters and channels arereadily deformable. Other regions of the pouch 10 may be made of a rigidmaterial or may be reinforced with a rigid material.

Illustratively, a plastic film is used for pouch 10. A sheet of metal,illustratively aluminum, or other suitable material, may be milled orotherwise cut, to create a die having a pattern of raised surfaces. Whenfitted into a pneumatic press (illustratively A-5302-PDS, JanesvilleTool Inc., Milton Wis.), illustratively regulated at an operatingtemperature of 195° C., the pneumatic press works like a printing press,melting the sealing surfaces of plastic film only where the die contactsthe film. Various components, such as PCR primers (illustrativelyspotted onto the film and dried), antigen binding substrates, magneticbeads, and zirconium silicate beads may be sealed inside variousblisters as the pouch 10 is formed. Reagents for sample processing canbe spotted onto the film prior to sealing, either collectively orseparately. In one embodiment, nucleotide tri-phosphates (NTPs) arespotted onto the film separately from polymerase and primers,essentially eliminating activity of the polymerase until the reaction ishydrated by an aqueous sample. If the aqueous sample has been heatedprior to hydration, this creates the conditions for a true hot-start PCRand reduces or eliminates the need for expensive chemical hot-startcomponents. This separate spotting is discussed further below, withrespect to FIG. 5 b, but it is understood that such spotting may be usedwith any of the embodiments discussed herein.

When pneumatic pressure is used to move materials within pouch 10, inone embodiment a “bladder” may be employed. The bladder assembly 710, aportion of which is shown in FIG. 2 a, may be manufactured in a processsimilar to that of making the pouch, but individual blisters in thebladder assembly 710 include pneumatic fittings (illustratively fitting724 a) allowing individual bladders within the bladder assembly 710 tobe pressurized by a compressed gas source. Because the bladder assemblyis subjected to compressed gas and may be used multiple times, thebladder assembly may be made from tougher or thicker material than thepouch. Alternatively, bladders may be formed from a series of platesfastened together with gaskets, seals, valves, and pistons. Otherarrangements are within the scope of this invention.

When pouch 10 is placed within the instrument, the pneumatic bladderassembly 710 is pressed against one face of the pouch 10, so that if aparticular bladder is inflated, the pressure will force the liquid outof the corresponding blister in the pouch 10. In addition to pneumaticbladders corresponding to many of the blisters of pouch 10, the bladderassembly may have additional pneumatic actuators, such as bladders orpneumatically-driven pistons, corresponding to various channels of pouch10. When activated, these additional pneumatic actuators form pinchvalves to pinch off and close the corresponding channels. To confineliquid within a particular blister of pouch 10, the pinch valvepneumatic actuators are inflated over the channels leading to and fromthe blister, such that the actuators function as pinch valves to pinchthe channels shut. Illustratively, to mix two volumes of liquid indifferent blisters, the pinch valve pneumatic actuator sealing theconnecting channel is depressurized, and the pneumatic bladders over theblisters are alternately pressurized, forcing the liquid back and forththrough the channel connecting the blisters to mix the liquid therein.The pinch valve pneumatic actuators may be of various shapes and sizesand may be configured to pinch off more than one channel at a time. Suchan illustrative pinch valve is illustrated in FIG. 1 as pinch valve 16,which may be used to close all injection ports. While pneumaticactuators are discussed herein, it is understood that other ways ofproviding pressure to the pouch are contemplated, including variouselectromechanical actuators such as linear stepper motors, motor-drivencams, rigid paddles driven by pneumatic, hydraulic or electromagneticforces, rollers, rocker-arms, and in some cases, cocked springs. Inaddition, there are a variety of methods of reversibly or irreversiblyclosing channels in addition to applying pressure normal to the axis ofthe channel. These include kinking the bag across the channel,heat-sealing, rolling an actuator, and a variety of physical valvessealed into the channel such as butterfly valves and ball valves.Additionally, small Peltier devices or other temperature regulators maybe placed adjacent the channels and set at a temperature sufficient tofreeze the fluid, effectively forming a seal. Also, while the design ofFIG. 1 is adapted for an automated instrument featuring actuatorelements positioned over each of the blisters and channels, it is alsocontemplated that the actuators could remain stationary, and the pouchcould be transitioned in one or two dimensions such that a small numberof actuators could be used for several of the processing stationsincluding sample disruption, nucleic-acid capture, first andsecond-stage PCR, and other applications of the pouch such asimmuno-assay and immuno-PCR. Rollers acting on channels and blisterscould prove particularly useful in a configuration in which the pouch istranslated between stations. Thus, while pneumatic actuators are used inthe presently disclosed embodiments, when the term “pneumatic actuator”is used herein, it is understood that other actuators and other ways ofproviding pressure may be used, depending on the configuration of thepouch and the instrument.

With reference to FIG. 1, an illustrative sample pouch 10 configured fornucleic acid extraction and multiplex PCR is provided. The sample enterspouch 10 via sample injection port 12 in fitment 90. Injection port 12may be a frangible seal, a one-way valve, or other entry port. Vacuumfrom inside pouch 10 may be used to draw the sample into pouch 10, asyringe or other pressure may be used to force the sample into pouch 10,or other means of introducing the sample into pouch 10 via injector port12 may be used. The sample travels via channel 14 to the three-lobedblister 22 of the cell lysis zone 20, wherein cells in the sample arelysed. Once the sample enters three-lobed blister 22, pinch valve 16 isclosed. Along with pinch valve 36, which may have been already closed,the closure of pinch valve 16 seals the sample in three-lobed blister22. It is understood that cell lysis may not be necessary with everysample. For such samples, the cell lysis zone may be omitted or thesample may be moved directly to the next zone. However, with manysamples, cell lysis is needed. In one embodiment, bead-milling is usedto lyse the cells.

Bead-milling, by shaking or vortexing the sample in the presence oflysing particles such as zirconium silicate (ZS) beads 34, is aneffective method to form a lysate. It is understood that, as usedherein, terms such as “lyse,” “lysing,” and “lysate” are not limited torupturing cells, but that such terms include disruption of non-cellularparticles, such as viruses. FIG. 2 displays one embodiment of a celllysis zone 20, where convergent flow creates high velocity bead impacts,to create lysate. Illustratively, the two lower lobes 24, 26 ofthree-lobed blister 22 are connected via channel 30, and the upper lobe28 is connected to the lower lobes 24, 26 at the opposing side 31 ofchannel 30. FIG. 2 a shows a counterpart portion of the bladder assembly710 that would be in contact with the cell lysis zone 20 of the pouch10. When pouch 10 is placed in an instrument, adjacent each lobe 24, 26,28 on pouch 10 is a corresponding pneumatic bladder 724, 726, 728 in thebladder assembly 710. It is understood that the term “adjacent,” whenreferring to the relationship between a blister or channel in a pouchand its corresponding pneumatic actuator, refers to the relationshipbetween the blister or channel and the corresponding pneumatic actuatorwhen the pouch is placed into the instrument. In one embodiment, thepneumatic fittings 724 a, 726 a of the two lower pneumatic bladders 724,726 adjacent lower lobes 24, 26 are plumbed together. The pneumaticfittings 724 a, 726 a and the pneumatic fitting 728 a of upper pneumaticbladder 728 adjacent upper lobe 28 are plumbed to the opposing side ofan electrically actuated valve configured to drive a double-actingpneumatic cylinder. Thus configured, pressure is alternated between theupper pneumatic bladder 728 and the two lower pneumatic bladders 724,726. When the valve is switched back and forth, liquid in pouch 10 isdriven between the lower lobes 24, 26 and the upper lobe 28 through anarrow nexus 32 in channel 30. As the two lower lobes 24, 26 arepressurized at the same time, the flow converges and shoots into theupper lobe 28. Depending on the geometry of the lobes, the collisionvelocity of beads 34 at the nexus 32 may be at least about 12 m/sec,providing high-impact collisions resulting in lysis. The illustrativethree-lobed system allows for good cell disruption and structuralrobustness, while minimizing size and pneumatic gas consumption. WhileZS beads are used as the lysing particles, it is understood that thischoice is illustrative only, and that other materials and particles ofother shapes may be used. It is also understood that otherconfigurations for cell lysis zone 20 are within the scope of thisinvention.

While a three-lobed blister is used for cell lysis, it is understoodthat other multi-lobed configurations are within the scope of thisinvention. For instance, a four-lobed blister, illustratively in acloverleaf pattern, could be used, wherein the opposite blisters arepressurized at the same time, forcing the lysing particles toward eachother, and then angling off to the other two lobes, which then may bepressurized together. Such a four-lobed blister would have the advantageof having high-velocity impacts in both directions. Further, it iscontemplated that single-lobed blisters may be used, wherein the lysingparticles are moved rapidly from one portion of the single-lobed blisterto the other. For example, pneumatic actuators may be used to close offareas of the single-lobed blister, temporarily forming a multi-lobedblister in the remaining areas. Other actuation methods may also be usedsuch as motor, pneumatic, hydraulic, or electromagnetically-drivenpaddles acting on the lobes of the device. Rollers or rotary paddles canbe used to drive fluid together at the nexus 32 of FIG. 2,illustratively if a recirculation means is provided between the upperand lower lobes and the actuator provides peristaltic pumping action.Other configurations are within the scope of this invention.

It may also be possible to move the sample and lysing particles quicklyenough to effect lysis within a single-lobed lysis blister withouttemporarily forming a multi-lobed blister. In one such alternativeembodiment, as shown in FIG. 2 b, vortexing may be achieved by impactingthe pouch with rotating blades or paddles 21 attached to an electricmotor 19. The blades 21 may impact the pouch at the lysis blister or mayimpact the pouch near the lysis blister, illustratively at an edge 17adjacent the lysis blister. In such an embodiment, the lysis blister maycomprise one or more blisters. FIG. 12 shows an embodiment comprisingone such lysis blister 522. FIG. 13 shows a bead beating motor 19,comprising blades 21, that may be mounted on a first side 811 of secondsupport member 804, of instrument 800 shown in FIG. 8. It is understood,however, that motor 19 may be mounted on first support member 802 or onother structure of instrument 800.

FIG. 2 a also shows pneumatic bladder 716 with pneumatic fitting 716 a,and pneumatic bladder 736 with pneumatic fitting 736 a. When the pouch10 is placed in contact with bladder assembly 710, bladder 716 lines upwith channel 12 to complete pinch valve 16. Similarly, bladder 736 linesup with channel 38 to complete pinch valve 36. Operation of pneumaticbladders 716 and 736 allow pinch valves 16 and 36 to be opened andclosed.

While only the portion of bladder assembly 710 adjacent the cell lysiszone is shown, it is understood that bladder assembly 710 would beprovided with similar arrangements of pneumatic blisters to control themovement of fluids throughout the remaining zones of pouch 10.

Other prior art instruments teach PCR within a sealed flexiblecontainer. See, e.g., U.S. Pat. Nos. 6,645,758 and 6,780,617, andco-pending U.S. patent application Ser. No. 10/478,453, hereinincorporated by reference. However, including the cell lysis within thesealed PCR vessel can improve ease of use and safety, particularly ifthe sample to be tested may contain a biohazard. In the embodimentsillustrated herein, the waste from cell lysis, as well as that from allother steps, remains within the sealed pouch. However, it is understoodthat the pouch contents could be removed for further testing.

Once the cells are lysed, pinch valve 36 is opened and the lysate ismoved through channel 38 to the nucleic acid preparation zone 40, asbest seen in FIG. 3, after which, pinch valve 36 is closed, sealing thesample in nucleic acid preparation zone 40. In the embodimentillustrated in FIG. 3, purification of nucleic acids takes thebead-milled material and uses affinity binding to silica-basedmagnetic-beads 56, washing the beads with ethanol, and eluting thenucleic acids with water or other fluid, to purify the nucleic acid fromthe cell lysate. The individual components needed for nucleic acidextraction illustratively reside in blisters 44, 46, 48, which areconnected by channels 45, 47, 49 to allow reagent mixing. The lysateenters blister 44 from channel 38. Blister 44 may be provided withmagnetic beads 56 and a suitable binding buffer, illustratively ahigh-salt buffer such as that of 1-2-3™ Sample Preparation Kit (IdahoTechnology, Salt Lake City, Utah) or either or both of these componentsmay be provided subsequently through one or more channels connected toblister 44. The nucleic acids are captured on beads 56, pinch valve 53is then opened, and the lysate and beads 56 may be mixed by gentlepressure alternately on blisters 44 and 58 and then moved to blister 58via pneumatic pressure illustratively provided by a correspondingpneumatic bladder on bladder assembly 710. The magnetic beads 56 arecaptured in blister 58 by a retractable magnet 50, which is located inthe instrument adjacent blister 58, and waste may be moved to a wastereservoir or may be returned to blister 44 by applying pressure toblister 58. Pinch valve 53 is then closed. The magnetic beads 56 arewashed with ethanol, isopropanol, or other organic or inorganic washsolution provided from blister 46, upon release of pinch valve 55.Optionally, magnet 50 may be retracted allowing the beads to be washedby providing alternate pressure on blisters 46 and 58. The beads 56 areonce again captured in blister 58 by magnet 50, and the non-nucleic acidportion of the lysate is washed from the beads 56 and may be moved backto blister 46 and secured by pinch valve 55 or may be washed away viaanother channel to a waste reservoir. Once the magnetic beads arewashed, pinch valve 57 is opened, releasing water (illustrativelybuffered water) or another nucleic acid eluant from blister 48. Onceagain, the magnet 50 may be retracted to allow maximum mixing of waterand beads 56, illustratively by providing alternate pressure on blisters48 and 58. The magnet 50 is once again deployed to collect beads 56.Pinch valve 59 is released and the eluted nucleic acid is moved viachannel 52 to first-stage amplification zone 60. Pinch valve 59 is thenclosed, thus securing the sample in first-stage amplification zone 60.

It is understood that the configuration for the nucleic acid preparationzone 40, as shown in FIG. 3 and described above, is illustrative only,and that various other configurations are possible within the scope ofthe present disclosure.

The ethanol, water, and other fluids used herein may be provided to theblisters in various ways. The fluids may be stored in the blisters, thenecks of which may be pinched off by various pinch valves or frangibleportions that may be opened at the proper time in the sample preparationsequence. Alternatively, fluid may be stored in reservoirs in the pouchas shown pouch 110 in FIG. 5, or in the fitment as discussed withrespect to pouch 210 of FIG. 6, and moved via channels, as necessary. Instill another embodiment, the fluids may be introduced from an externalsource, as shown in FIG. 1, especially with respect to ethanol injectionports 41, 88 and plungers 67, 68, 69. Illustratively, plungers 67, 68,69 may inserted into fitment 90, illustratively of a more rigidmaterial, and may provide a measured volume of fluid upon activation ofthe plunger, as in U.S. Pat. No. 8,409,508, herein incorporated byreference. Alternatively, plunger may be a softer material and thefitment may be the more rigid material. The measured volume may be thesame or different for each of the plungers. Finally, in yet anotherembodiment, the pouch may be provided with a measured volume of thefluid that is stored in one or more blisters, wherein the fluid iscontained within the blister, illustratively provided in a small sealedpouch within the blister, effectively forming a blister within theblister. At the appropriate time, the sealed pouch may then be ruptured,illustratively by pneumatic pressure, thereby releasing the fluid intothe blister of the pouch. The instrument may also be configured theprovide some or all of the reagents directly through liquid contactsbetween the instrument and the fitment or pouch material provided thatthe passage of fluid is tightly regulated by a one-way valve to preventthe instrument from becoming contaminated during a run. Further, it willoften be desirable for the pouch or its fitment to be sealed afteroperation to prohibit contaminating DNA to escape from the pouch.Various means are known to provide reagents on demand such as syringepumps, and to make temporary fluid contact with the fitment or pouch,such as barbed fittings or o-ring seals. It is understood that any ofthese methods of introducing fluids to the appropriate blister may beused with any of the embodiments of the pouch as discussed herein, asmay be dictated by the needs of a particular application.

As discussed above, nested PCR involves target amplification performedin two stages. In the first-stage, targets are amplified, illustrativelyfrom genomic or reverse-transcribed template. The first-stageamplification may be terminated prior to plateau phase, if desired. Inthe secondary reaction, the first-stage amplicons may be diluted and asecondary amplification uses the same primers or illustratively usesnested primers hybridizing internally to the primers of the first-stageproduct. Advantages of nested PCR include: a) the initial reactionproduct forms a homogeneous and specific template assuring high fidelityin the secondary reaction, wherein even a relatively low-efficiencyfirst-stage reaction creates adequate template to support robustsecond-stage reaction; b) nonspecific products from the first-stagereaction do not significantly interfere with the second stage reaction,as different nested primers are used and the original amplificationtemplate (illustratively genomic DNA or reverse-transcription product)may be diluted to a degree that eliminates its significance in thesecondary amplification; and c) nested PCR enables higher-order reactionmultiplexing. First-stage reactions can include primers for severalunique amplification products. These products are then identified in thesecond-stage reactions. However, it is understood that first-stagemultiplex and second-stage singleplex is illustrative only and thatother configurations are possible. For example, the first-stage mayamplify a variety of different related amplicons using a single pair ofprimers, and second-stage may be used to target differences between theamplicons, illustratively using melting curve analysis.

Turning back to FIG. 1, the nucleic acid sample enters the first-stageamplification zone 60 via channel 52 and is delivered to blister 61. APCR mixture, including a polymerase (illustratively a Taq polymerase),dNTPs, and primers, illustratively a plurality of pairs of primers formultiplex amplification, may be provided in blister 61 or may beintroduced into blister 61 via various means, as discussed above.Alternatively, dried reagents may be spotted onto the location ofblister 61 upon assembly of pouch 10, and water or buffer may beintroduced to blister 61, illustratively via plunger 68, as shown inFIG. 1. As best seen in FIG. 4, the sample is now secured in blister 61by pinch valves 59 and 72, and is thermocycled between two or moretemperatures, illustratively by heat blocks or Peltier devices that arelocated in the instrument and configured to contact blister 61. However,it is understood that other means of heating and cooling the samplecontained within blister 61, as are known in the art, are within thescope of this invention. Non-limiting examples of alternativeheating/cooling devices for thermal cycling include having a air-cycledblister within the bladder, in which the air in the pneumatic blisteradjacent blister 61 is cycled between two or more temperatures; ormoving the sample to temperature zones within the blister 61,illustratively using a plurality of pneumatic presses, as in U.S. patentapplication Ser. No. 10/478,453, herein incorporated by reference, or bytranslating pouch 10 on an axis or providing pouch 10 with a rotarylayout and spinning pouch 10 to move the contents between heat zones offixed temperature.

Nucleic acids from pathogens are often co-isolated with considerablequantities of host nucleic acids. These host-derived nucleic acids ofteninteract with primers, resulting in amplification of undesired productsthat then scavenge primers, dNTPs, and polymerase activity, potentiallystarving a desired product of resources. Nucleic acids from pathogenicorganisms are generally of low abundance, and undesired product is apotential problem. The number of cycles in the first-stage reaction ofzone 60 may be optimized to maximize specific products and minimizenon-specific products. It is expected that the optimum number of cycleswill be between about 10 to about 30 cycles, illustratively betweenabout 15 to about 20 cycles, but it is understood that the number ofcycles may vary depending on the particular target, host, and primersequence.

Following the first-stage multiplex amplification, the first-stageamplification product is diluted, illustratively in incomplete PCRmaster mix, before fluidic transfer to secondary reaction sites.

FIG. 4 shows an illustrative embodiment for diluting the sample in threesteps. In the first step, pinch valve 72 is opened and the sampleundergoes a two-fold dilution by mixing the sample in blister 61 with anequal volume of water or buffer from blister 62, which is provided toblister 62, as well as blisters 64 and 66, as discussed above. Squeezingthe volume back and forth between blisters 61, 62 provides thoroughmixing. As above, mixing may be provided by pneumatic bladders providedin the bladder 710 and located adjacent blisters 61, 62. The pneumaticbladders may be alternately pressurized, forcing the liquid back andforth. During mixing, a pinch valve 74 prevents the flow of liquid intothe adjacent blisters. At the conclusion of mixing, a volume of thediluted sample is captured in region 70, and pinch valve 72 is closed,sealing the diluted sample in region 70. Pinch valve 74 is opened andthe sample is further diluted by water or buffer provided in either orboth of blisters 63, 64. As above, squeezing the volume back and forthbetween blisters 63, 64 provides mixing. Subsequently, pinch valve 74 isclosed, sealing a further diluted volume of sample in region 71. Finaldilution takes place illustratively by using buffer or water provided ineither or both of blisters 65, 66, with mixing as above. Illustrativelythis final dilution takes place using an incomplete PCR master mix(e.g., containing all PCR reagents except primers) as the fluid.Optional heating of the contents of blister 66 prior to second-stageamplification can provide the benefits of hot-start amplificationwithout the need for expensive antibodies or enzymes. It is understood,however, that water or other buffer may be used for the final dilution,with additional PCR components provided in second-stage amplificationzone 80. While the illustrative embodiment uses three dilution stages,it is understood that any number of dilution stages may be used, toprovide a suitable level of dilution. It is also understood that theamount of dilution can be controlled by adjusting the volume of thesample captured in regions 70 and 71, wherein the smaller the amount ofsample captured in regions 70 and 71, the greater the amount of dilutionor wherein additional aliquots captured in region 70 and/or region 71 byrepeatedly opening and closing pinch valves 72 and 74 and/or pinchvalves 74 and 76 may be used to decrease the amount of dilution. It isexpected that about 10⁻² to about 10⁻⁵ dilution would be suitable formany applications.

Success of the secondary PCR reactions is dependent upon templategenerated by the multiplex first-stage reaction. Typically, PCR isperformed using DNA of high purity. Methods such as phenol extraction orcommercial DNA extraction kits provide DNA of high purity. Samplesprocessed through the pouch 10 may require accommodations be made tocompensate for a less pure preparation. PCR may be inhibited bycomponents of biological samples, which is a potential obstacle.Illustratively, hot-start PCR, higher concentration of taq polymeraseenzyme, adjustments in MgCl₂ concentration, adjustments in primerconcentration, and addition of adjuvants (such as DMSO, TMSO, orglycerol) optionally may be used to compensate for lower nucleic acidpurity. While purity issues are likely to be more of a concern withfirst-stage amplification, it is understood that similar adjustments maybe provided in the second-stage amplification as well.

While dilution and second-stage sample preparation are accomplished inthe illustrative embodiment by retaining a small amount of amplifiedsample in the blisters and channels of the first-stage PCR portion ofthe pouch, it is understood that these processes may also be performedin other ways. In one such illustrative example, pre-amplified samplecan be captured in a small cavity in a member, illustratively atranslating or rotating member, able to move a fixed volume of samplefrom the first to the second-stage PCR reagent. A one microliterfraction of the pre-amplified sample, mixed with 100 microliters offresh PCR reagent would yield a one-hundred-fold reduction inconcentration. It is understood that this dilution is illustrative only,and that other volumes and dilution levels are possible. This approachcould be accomplished by forcing the first-stage amplification productinto the rigid fitment where it contacts one of the plungers 68 or 69 ofFIG. 1. In such an embodiment, the plunger would be configured to carrya small fraction of the sample into contact with the adjacent dilutionbuffer or second-stage PCR buffer. Similarly a sliding element could beused to carry a small amount of the first-stage amplification productinto contact with the second-stage reaction mix while maintaining a sealbetween the stages, and containing the amplified sample within the rigidfitment 90.

Subsequent to first-stage PCR and dilution, channel 78 transfers thesample to a plurality of low volume blisters 82 for secondary nestedPCR. In one illustrative embodiment, dried primers provided in thesecond-stage blisters are resuspended by the incoming aqueous materialto complete the reaction mixture. Optionally, fluorescent dyes such asLCGreen® Plus (Idaho Technology, Salt Lake City, Utah) used fordetection of double-stranded nucleic acid may be provided in eachblister or may be added to the incomplete PCR master mix provided at theend of the serial dilution, although it is understood that LCGreen® Plusis illustrative only and that other dyes are available, as are known inthe art. In another optional embodiment, dried fluorescently labeledoligonucleotide probes configured for each specific amplicon may beprovided in each respective second-stage blister, along with therespective dried primers. Further, while pouch 10 is designed to containall reactions and manipulations within, to reduce contamination, in somecircumstances it may be desirable to remove the amplification productsfrom each blister 82 to do further analysis. Other means for detectionof the second-stage amplicon, as are known in the art, are within thescope of this invention. Once the sample is transferred to blisters 82,pinch valves 84 and 86 are activated to close off blisters 82. Eachblister 82 now contains all reagents needed for amplification of aparticular target. Illustratively, each blister may contain a uniquepair of primers, or a plurality of blisters 82 may contain the sameprimers to provide a number of replicate amplifications.

It is noted that the embodiments disclosed herein use blisters for thesecond-stage amplification, wherein the blisters are formed of the sameor similar plastic film as the rest of the flexible portion. However, inmany embodiments, the contents of the second-stage blisters are neverremoved from the second-stage blisters, and, therefore, there is no needfor the second-stage reaction to take place in flexible blisters. It isunderstood that the second-stage reaction may take place in a pluralityof rigid, semi-rigid, or flexible chambers that are fluidly connected tothe blisters. The chambers could be sealed as in the present example byplacing pressure on flexible channels that connect the chambers, or maybe sealed in other ways, illustratively by heat sealing or use ofone-way valves. Various embodiments discussed herein include blistersprovided solely for the collection of waste. Since the waste may neverbe removed, waste could be collected in rigid, semi-rigid, or flexiblechambers.

It is within the scope of this invention to do the second-stageamplification with the same primers used in the first-stageamplification (see U.S. Pat. No. 6,605,451). However, it is oftenadvantageous to have primers in second-stage reactions that are internalto the first-stage product such that there is no or minimal overlapbetween the first- and second-stage primer binding sites. Dilution offirst-stage product largely eliminates contribution of the originaltemplate DNA and first-stage reagents to the second-stage reaction.Furthermore, illustratively, second-stage primers with a Tm higher thanthose used in the first-stage may be used to potentiate nestedamplification. Primer may be designed to avoid significant hairpins,hetero/homo-dimers and undesired hybridization. Because of the nestedformat, second-stage primers tolerate deleterious interactions far moreso than primers used to amplify targets from genomic DNA in a singlestep. Optionally, hot-start is used on second-stage amplification.

If a fluorescent dye is included in second-stage amplification,illustratively as a dsDNA binding dye or as part of a fluorescent probe,as are known in the art, optics provided may be used to monitoramplification of one or more of the samples. Optionally, analysis of theshape of the amplification curve may be provided to call each samplepositive or negative. Illustrative methods of calling the sample arediscussed in U.S. Pat. No. 6,730,501, herein incorporated by reference.Alternatively, methods employing a crossing threshold may be used. Acomputer may be provided externally or within the instrument and may beconfigured to perform the methods and call the sample positive ornegative based upon the presence or absence of second-stageamplification and may provide quantitative information about thestarting template concentration by comparing characteristic parametersof the amplification curve (such as crossing threshold) to standardcurves, or relative to other amplification curves within the run. It isunderstood, however, that other methods, as are known in the art, may beused to call each sample. Other analyses may be performed on thefluorescent information. One such non-limiting example is the use ofmelting curve analysis to show proper melting characteristics (e.g. Tm,melt profile shape) of the amplicon. The optics provided may beconfigured to capture images of all blisters 82 at once, or individualoptics may be provided for each individual blister. Other configurationsare within the scope of this invention.

FIG. 5 shows an alternative pouch 110. In this embodiment, variousreagents are loaded into pouch 110 via fitment 190. FIG. 5 a shows across-section of fitment 190 with one of a plurality of plungers 168. Itis understood that, while FIG. 5 a shows a cross-section through entrychannel 115 a, as shown in the embodiment of FIG. 5, there are 12 entrychannels present (entry channel 115 a through 115 l), each of which mayhave its own plunger 168 for use in fitment 190, although in thisparticular configuration, entry channels 115 c, 115 f, and 115 i are notused. It is understood that a configuration having 12 entry channels isillustrative only, and that any number of entry channels and associatedplungers may be used.

In the illustrative embodiment, an optional vacuum port 142 of fitment190 is formed through a first surface 194 of fitment 190 to communicatewith chamber 192. Optional vacuum port 142 may be provided forcommunication with a vacuum or vacuum chamber (not shown) to draw outthe air from within pouch 110 to create a vacuum within chamber 192 andthe various blisters and chambers of pouch 110. Plunger 168 is theninserted far enough into chamber 192 to seal off vacuum port 142.Chamber 192 is illustratively provided under a predetermined amount ofvacuum to draw a desired volume of liquid into chamber 192 upon use.Additional information on preparing chamber 192 may be found in U.S.Pat. No. 8,409,508, already incorporated by reference.

Illustrative fitment 190 further includes an injection port 141 formedin the second surface 195 of fitment 190. Illustratively, injection port141 is positioned closer to the plastic film portion of pouch 110 thanvacuum port 142, as shown in FIG. 5 a, such that the plunger 168 isinserted far enough to seal off vacuum port 142, while still allowingaccess to chamber 192 via injection port 141. As shown, second surface119 of plastic film portion 117 provides a penetrable seal 139 toprevent communication between chamber 192 and the surrounding atmospherevia injection port 141. However, it is understood that second surface119 optionally may not extend to injection port 141 and various otherseals may be employed. Further, if another location for the seal isdesired, for example on a first surface 194 of fitment 190, injectionport 141 may include a channel to that location on fitment 190. U.S.patent application Ser. No. 10/512,255, already incorporated byreference, shows various configurations where the seal is locatedremotely from the injection port, and the seal is connected to thechamber via a channel. Also, U.S. Pat. No. 8,409,508 discloses variousconfigurations where channels connect a single seal to multiplechambers. Variations in seal location, as well as connection of a singleinjection port to multiple chambers, are within the scope of thisinvention. Optionally, seal 139 may be frangible and may be broken uponinsertion of a cannula (not shown), to allow a fluid sample from withinthe cannula to be drawn into or forced into chamber 192.

The illustrative plunger 168 of the pouch assembly 110 is cylindrical inshape and has a diameter of approximately 5 mm to be press-fit intochamber 192. Plunger 168 includes a first end portion 173 and anopposite second end portion 175. As shown in FIG. 5 a, a notch 169 ofplunger 168 is formed in second end portion 175. In use, second endportion 175 is inserted part way into chamber 192, and notch 169 may bealigned with injection port 141 to allow a fluid sample to be drawn intoor injected into chamber 192, even when plunger 168 is inserted farenough that plunger 168 would otherwise be blocking injection port 141.Illustratively, a fluid is placed in a container (not shown) with asyringe having a cannulated tip that can be inserted into injection port141 to puncture seal 139 therein. In using an air-evacuated pouchassembly 110, when seal 139 is punctured, the fluid is withdrawn fromthe container due to the negative pressure within chamber 192 relativeto ambient air pressure. Fluid then passes through port 141 to fillchamber 192. At this point, the fluid usually does not flow into theplastic film portion 117 of pouch 110. Finally, the plunger 168 isinserted into chamber 192 such that second end portion 175 of plunger168 approaches the bottom 191 of chamber 192, to push a measured amountof the reagent or sample into the plastic film portion 117. As shown,plunger 168 is configured such that upon full insertion, second endportion 175 does not quite meet bottom 191 of chamber 192. The remainingspace is useful in trapping bubbles, thereby reducing the number ofbubbles entering plastic film portion 117. However, in some embodimentsit may be desirable for second end portion 175 to meet bottom 191 uponfull insertion of plunger 168. In the embodiment shown in FIG. 5, entrychannels 115 a, 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and 115l all lead to reaction zones or reservoir blisters. It is understoodthat full insertion of the plunger associated with entry channel 115 awould force a sample into three-lobed blister 122, full insertion of theplunger associated with entry channel 115 b would force a reagent intoreservoir blister 101, full insertion of the plunger associated withentry channel 115 d would force a reagent into reservoir blister 102,full insertion of the plunger associated with entry channel 115 e wouldforce a reagent into reservoir blister 103, full insertion of theplunger associated with entry channel 115 g would force a reagent intoreservoir blister 104, full insertion of the plunger associated withentry channel 115 h would force a reagent into reservoir blister 105,full insertion of the plunger associated with entry channel 115 j wouldforce a reagent into reservoir blister 106, full insertion of theplunger associated with entry channel 115 k would force a reagent intoreservoir blister 107, and full insertion of the plunger associated withentry channel 115 l would force a reagent into reservoir blister 108.

If a plunger design is used including notch 169 as illustrated in theembodiment shown in FIG. 5 a, the plunger 168 may be rotated prior tobeing lowered, so as to offset notch 169 and to close off injection port141 from communication with chamber 192, to seal the contents therein.This acts to minimize any potential backflow of fluid through injectionport 141 to the surrounding atmosphere, which is particularly usefulwhen it is desired to delay in full insertion of the plunger. Althoughnotch 169 is shown and described above with respect to plunger 168, itis within the scope of this disclosure to close off injection port 141soon after dispensing the fluid sample into the chamber 192 by othermeans, such as depressing plunger 168 toward the bottom of chamber 192,heat sealing, unidirectional valves, or self-sealing ports, for example.If heat sealing is used as the sealing method, a seal bar could beincluded in the instrument such that all chambers are heat sealed uponinsertion of the pouch into the instrument.

In the illustrative method, the user injects the sample into theinjection port 141 associated with entry channel 115 a, and water intothe various other injection ports. The water rehydrates reagents thathave been previously freeze-dried into chambers 192 associated with eachof entry channels 115 b, 115 d, 115 e, 115 g, 115 h, 115 j, 115 k, and115 l. The water may be injected through one single seal and then bedistributed via a channel to each of the chambers, as shown in FIG. 6below, or the water could be injected into each chamber independently.Alternatively, rather than injecting water to rehydrate dried reagents,wet reagents such as lysis reagents, nucleic acid extraction reagents,and PCR reagents may be injected into the appropriate chambers 192 ofthe fitment 190.

Upon activation of the plunger 168 associated with entry channel 115 a,the sample is forced directly into three-lobed blister 122 via channel114. The user also presses the remaining plungers 168, forcing thecontents out of each of the chambers 192 in fitment 190 and intoreservoir blisters 101 through 108. At this point, pouch 110 is loadedinto an instrument for processing. While instrument 800, shown in FIG.8, is configured for the pouch 210 of FIG. 6, it is understood thatmodification of the configuration of the bladders of instrument 800would render instrument 800 suitable for use with pouches 110 and 510,or with pouches of other configurations.

In one illustrative example, upon depression of the plungers 168,reservoir blister 101 now contains DNA-binding magnetic beads inisopropanol, reservoir blister 102 now contains a first wash solution,reservoir blister 103 now contains a second wash solution, reservoirblister 104 now contains a nucleic acid elution buffer, reservoirblister 105 now contains first-stage PCR reagents, including multiplexedfirst-stage primers, reservoir blister 106 now contains second-stage PCRreagents without primers, reservoir blister 107 now contains negativecontrol PCR reagents without primers and without template, and reservoirblister 108 now contains positive control PCR reagents with template.However, it is understood that these reagents are illustrative only, andthat other reagents may be used, depending upon the desired reactionsand optimization conditions.

Once pouch 110 has been placed into instrument 800 and the sample hasbeen moved to three-lobed blister 122, the sample may be subjected todisruption by agitating the sample with lysing particles such as ZS orceramic beads. The lysing particles may be provided in three-lobedblister 122, or may be injected into three-lobed blister 122 along withthe sample. The three-lobed blister 122 of FIG. 5 is operated in muchthe same way as three-lobed blister 22 of FIG. 1, with the two lowerlobes 124, 126 pressurized together, and pressure is alternated betweenthe upper lobe 128 and the two lower lobes 124, 126. However, asillustrated, lower lobes 124, 126 are much more rounded than lower lobes24, 26, allowing for a smooth flow of beads to channel 130 and allowingfor high-speed collisions, even without the triangular flow separator atnexus 32. As with three-lobed blister 22, three-lobed blister 122 ofFIG. 5 allows for effective lysis or disruption of microorganisms,cells, and viral particles in the sample. It has been found that achannel 130 having a width of about 3-4 mm, and illustratively about 3.5mm, remains relatively clear of beads during lysis and is effective inproviding for high-velocity collisions.

After lysis, nucleic-acid-binding magnetic beads are injected into upperlobe 128 via channel 138 by pressurizing a bladder positioned overreservoir blister 101. The magnetic beads are mixed, illustratively moregently than with during lysis, with the contents of three-lobed blister122, and the solution is incubated, illustratively for about 1 minute,to allow nucleic acids to bind to the beads.

The solution is then pumped into the “figure 8” blister 144 via channel143, where the beads are captured by a retractable magnet housed in theinstrument, which is illustratively pneumatically driven. The beadcapture process begins by pressurizing all lobes 124, 126, and 128 ofthe bead milling apparatus 122. This forces much of the liquid contentsof 122 through channel 143 and into blister 144. A magnet is broughtinto contact with the lower portion 144 b of blister 144 and the sampleis incubated for several seconds to allow the magnet to capture thebeads from the solution, then the bladders adjacent to blister 122 aredepressurized, the bladders adjacent blister portions 144 a and 144 bare pressurized, and the liquid is forced back into blister 122. Sincenot all of the beads are captured in a single pass, this process may berepeated up to 10 times to capture substantially all of the beads inblister 144. Then the liquid is forced out of blister 144, leavingbehind only the magnetic beads and the captured nucleic acids, and washreagents are introduced into blister 144 in two successive washes (fromreservoir blisters 102 and 103 via channels 145 and 147, respectively).In each wash, the bladder positioned over the reservoir blistercontaining the wash reagent is pressurized, forcing the contents intoblister 144. The magnet is withdrawn and the pellet containing themagnetic beads is disrupted by alternatively pressurizing each of twobladders covering each lobe 144 a and 144 b of blister 144. When theupper lobe 144 a is compressed, the liquid contents are forced into thelower lobe 144 b, and when the lower lobe 144 b is compressed, thecontents are forced into the upper lobe 144 a. By agitating the solutionin blister 144 between upper lobe 144 a and lower lobe 144 b, themagnetic beads are effectively washed of impurities. A balance ismaintained between inadequate agitation, leaving the pellet of beadsundisturbed, and excessive agitation, potentially washing the nucleicacids from the surface of the beads and losing them with the washreagents. After each wash cycle, the magnetic beads are captured via themagnet in blister 144 and the wash reagents are illustratively forcedinto three-lobed blister 122, which now serves as a waste receptacle.However, it is understood that the used reservoir blisters may alsoserve as waste receptacles, or other blisters may be providedspecifically as waste receptacles.

Nucleic acid elution buffer from reservoir blister 104 is then injectedvia channel 149 into blister 144, the sample is once again agitated, andthe magnetic beads are recaptured by employment of the magnet. The fluidmixture in blister 144 now contains nucleic acids from the originalsample. Pressure on blister 144 moves the nucleic acid sample to thefirst stage PCR blister 161 via channel 152, where the sample is mixedwith first-stage PCR master mix containing multiple primer sets, the PCRmaster mix provided from reservoir blister 105 via channel 162. Ifdesired, the sample and/or the first-stage PCR master mix may be heatedprior to mixing, to provide advantages of hot start. Optionally,components for reverse transcription of RNA targets may be providedprior to first-stage PCR. Alternatively, an RT enzyme, illustratively athermostable RT enzyme may be provided in the first-stage PCR master mixto allow for contemporaneous reverse transcription of RNA targets. It isunderstood that an RT enzyme may be present in the first-stage PCRmixture in any of the embodiments disclosed herein. As will be seenbelow, pouch 110 of FIG. 5 is configured for up to 10 primer sets, butit is understood that the configuration may be altered and any number ofprimer sets may be used. A bladder positioned over blister 161 ispressurized at low pressure, to force the contents of blister 161 intointimate contact with a heating/cooling element, illustratively aPeltier element, on the other side of blister 161. The pressure onblister 161 should be sufficient to assure good contact with theheating/cooling element, but should be gentle enough such that fluid isnot forced from blister 161. The heating/cooling element is temperaturecycled, illustratively between about 60° C. to about 95° C.

Illustratively, temperature cycling is performed for about 15-20 cycles,resulting in amplification of one or more nucleic acid targets present.Also illustratively, temperature cycling ceases prior to plateau phase,and may cease in log phase or even prior to log phase. In one example,it may be desirable merely to enrich the sample with the desiredamplicons, without reaching minimal levels of detection. See U.S. Pat.No. 6,605,451, herein incorporated by reference.

The amplified sample is optionally then diluted by forcing most thesample back into blister 144 via channel 152, leaving only a smallamount (illustratively about 1 to 5%) of the amplified sample in blister161, and second-stage PCR master mix is provided from reservoir blister106 via channel 163. The sample is thoroughly mixed illustratively bymoving it back and forth between blisters 106 and 161 via channel 163.If desired, the reaction mixture may be heated to above extensiontemperature, illustratively at least 60° C., prior to second-stageamplification. The sample is then forced through channel 165 into anarray of low volume blisters 182 in the center of second-stageamplification zone 180. Each of the ten illustrative low volume blisters182 may contain a different primer pair, either essentially the same asone of the primer pairs in the first-stage amplification, or “nested”within the first-stage primer pair to amplify a shortened amplicon. Theprimers, now hydrated by the sample, complete the amplification mixture.Positive and negative control samples are also introduced bypressurizing the contents of reservoir blisters 107 and 108,respectively, forcing PCR master mix either without target DNA fromreservoir blister 107 via channel 166, or with control DNA fromreservoir blister 108, via channel 167. As illustrated, there are fiveeach of positive control blisters 183 and negative control blisters 181,which may be multiplexed 2-fold to provide the necessary controls forten different second-stage amplification reactions. It is understoodthat this configuration is illustrative only and that any number ofsecond-stage blisters may be provided.

Illustratively, the PCR master mix used for second-stage amplificationlacks the primers, but is otherwise complete. However, an “incomplete”PCR master mix may lack other PCR components as well. In one example,the second-stage PCR master mix is water or buffer only, which is thenmixed with the optionally diluted first-stage PCR amplification product.This mixture is moved to the small-volume PCR reaction blisters, whereall of the remaining components have been previously provided. Ifdesired, all of the remaining components may be mixed together andspotted as a single mixture into the small-volume PCR reaction blisters.Alternatively, as illustrated in FIG. 5 b, each of the components may bespotted onto a separate region of the small-volume PCR reaction blister182. As shown in FIG. 5 b, four regions are present, illustratively withdNTPs spotted at region 182 a, primers spotted at 182 b, polymerasespotted at 182 c, and a magnesium compound spotted at 182 d. By spottingthe components separately and heating the sample mixture prior torehydrating the components, nonspecific reactions can be minimized. Itis understood that any combination of components can be spotted thisway, and that this method of spotting components into one or moreregions of the blisters may be used with any embodiment of the presentinvention.

The channels 165, 166, and 167 leading to the small-volume PCR reactionblisters 181, 182, and 183 are sealed, and a pneumatic bladder gentlypresses the array against a heating/cooling element, illustratively aPeltier element, for thermal cycling. The cycling parameters may beindependently set for second-stage thermal cycling. Illustratively, thereactions are monitored by focusing an excitation source, illustrativelya blue light (450-490 nm), onto the array, and imaging the resultantfluorescent emissions, illustratively fluorescent emissions above 510nm.

In the above example, pinch valves are not discussed. However, it isunderstood that when it is desired to contain a sample in one of theblisters, pneumatic actuators positioned over channels leading to andfrom the particular blister are pressurized, creating pinch valves andclosing off the channels. Conversely, when it is desired to move asample from one of the blisters, the appropriate pneumatic actuator isdepressurized, allowing the sample flow through the channel.

The pouch described above in FIG. 5 includes reagent reservoir blisters101 through 108, in which the user injected reagents from the fitment190 into the reagent reservoir blisters 101 through 108 in the plasticfilm portion 117 of the pouch 110, illustratively prior to insertion ofpouch 110 into the instrument. While there are advantages to the use ofthe reagent reservoir blisters of FIG. 5, including having the abilityto maintain the contents of the various blisters at differenttemperatures, there are some disadvantages as well. Because the operatoris responsible for moving the reagents from the fitment 190 to thereservoir blisters 101 through 108, and because this is often doneoutside of the machine and thus without activated pinch valves, reagentscould occasionally leak from the reservoir blisters to the workingblisters. The reagents in reservoir blisters are exposed duringpreparation and loading. If they are pressed, squeezed, or even lightlybumped, the reagents may leak through available channels. If the loss ofreagents is substantial, the reaction may fail completely. Furthermore,during operation there may be some variability in the amount of reagentforced from the reservoir blisters 101 through 108, leading toinconsistent results. Automation of introduction of the reagents tofitment 190 and movement of the reagents from fitment 190 to reagentreservoir blisters 101 through 108 would solve many of these problems,and is within the scope of this invention.

The pouch 210 of FIG. 6 addresses many of these issues in a differentway, by using a direct-injection approach wherein the instrument itselfmoves the plungers 268, illustratively via pneumatic pistons, and forcesthe reagents into the various working blisters as the reagents areneeded. Rather than storing the reagents in reservoir blisters 101through 108 of FIG. 5, in the embodiment of FIG. 6 the reagents areintroduced into various chambers 292 of fitment 290 and are maintainedthere until needed. Pneumatic operation of piston 268 at the appropriatetime introduces a measured amount of the reagent to the appropriatereaction blister. In addition to addressing many of the above-mentionedissues, pouch 210 also has a much more compact shape, allowing for asmaller instrument design, and pouch 210 has shorter channels,permitting better fluid flow and minimizing reagent loss in channels.

In one illustrative embodiment of FIG. 6, a 300 μl mixture comprisingthe sample to be tested (100 μl) and lysis buffer (200 μl) is injectedinto injection port 241 a. Water is also injected into the fitment 290via seal 239 b, hydrating up to eleven different reagents, each of whichwere previously provided in dry form in chambers 292 b through 292 l viachannel 293 (shown in shadow). These reagents illustratively may includefreeze-dried PCR reagents, DNA extraction reagents, wash solutions,immunoassay reagents, or other chemical entities. For the example ofFIG. 6, the reagents are for nucleic acid extraction, first-stagemultiplex PCR, dilution of the multiplex reaction, and preparation ofsecond-stage PCR reagents, and control reactions. In the embodimentshown in FIG. 6, all that need be injected is the sample in port 241 aand water in port 241 b.

As shown in FIG. 6, water injected via seal 293 b is distributed tovarious chambers via channel 293. In this embodiment, only the sampleand water need be injected into pouch 210. It is understood, however,that water could be injected into each chamber 292 independently.Further, it is understood that, rather than providing dried reagents inthe various chambers 292 and hydrating upon injection of the water,specific wet reagents could be injected into each chamber, as desired.Additionally, it is understood that one or more of chambers 292 could beprovided with water only, and the necessary reagents may be provideddried in the appropriate reaction blisters. Various combinations of theabove, as dictated by the needs of the particular reaction, are withinthe scope of this invention.

As seen in FIG. 6, optional protrusions 213 are provided on bottomsurface 297 of fitment 290. As shown, protrusions 213 are located withintheir respective entry channels 215. However, other configurations arepossible. Protrusions 213 assist in opening entry channel 215 andprevent bottom surface 297 from engaging another flat surface in such away to pinch off entry channels 215 when plungers 268 are depressed,which helps prevent back-flow upon activation of the plungers 268. Suchprotrusions may be used on any of the various pouches according to thepresent invention. In embodiments wherein water is injected into thepouch to hydrate multiple dry reagents in multiple chambers in thefitment, a means of closing the channel between the injection port andthe many chambers is desired. If the channel is not closed, activationof each plunger may force some of the contents of its respective chamberback out into the channel, potentially contaminating neighboringchambers and altering the volumes contained in and delivered from thechamber. Several ways of closing this channel have been used, includingrotating a notched plunger 268 as discussed above, and heat-sealing theplastic film across the channel thereby closing the channel permanently,and applying pressure to the channel as a pinch valve. Other closuresmay also be used, such as valves built into the fitment, illustrativelyone-way valves.

After the fluids are loaded into chambers 292 and pouch 210 is loadedinto the instrument, plunger 268 a is depressed illustratively viaactivation of a pneumatic piston, forcing the balance of the sample intothree-lobed blister 220 via channel 214. As with the embodiments shownin FIGS. 1 and 5, the lobes 224, 226, and 228 of three-lobed blister 220are sequentially compressed via action bladders 824, 826, and 828 ofbladder assembly 810, shown in FIGS. 7-9, forcing the liquid through thenarrow nexus 232 between the lobes, and driving high velocitycollisions, shearing the sample and liberating nucleic acids,illustratively including nucleic acids from hard-to-open spores,bacteria, and fungi. Cell lysis continues for an appropriate length oftime, illustratively 0.5 to 10 minutes.

Once the cells have been adequately lysed, plunger 268 b is activatedand nucleic acid binding magnetic beads stored in chamber 292 b areinjected via channel 236 into upper lobe 228 of three-lobed blister 220.The sample is mixed with the magnetic beads and the mixture is allowedto incubate for an appropriate length of time, illustrativelyapproximately 10 seconds to 10 minutes.

The mixture of sample and beads are forced through channel 238 intoblister 244 via action of bladder 826, then through channel 243 and intoblister 246 via action of bladder 844, where a retractable magnet 850located in instrument 800 adjacent blister 245, shown in FIG. 8,captures the magnetic beads from the solution, forming a pellet againstthe interior surface of blister 246. A pneumatic bladder 846, positionedover blister 246 then forces the liquid out of blister 246 and backthrough blister 244 and into blister 222, which is now used as a wastereceptacle. However, as discussed above with respect to FIG. 5, otherwaste receptacles are within the scope of this invention. One ofplungers 268 c, 268 d, and 268 e may be activated to provide a washsolution to blister 244 via channel 245, and then to blister 246 viachannel 243. Optionally, the magnet 850 is retracted and the magneticbeads are washed by moving the beads back and forth from blisters 244and 246 via channel 243, by alternatively pressurizing bladders 844 and846. Once the magnetic beads are washed, the magnetic beads arerecaptured in blister 246 by activation of magnet 850, and the washsolution is then moved to blister 222. This process may be repeated asnecessary to wash the lysis buffer and sample debris from the nucleicacid-binding magnetic beads. Illustratively, three washes are done, oneeach using wash reagents in chambers 292 c, 292 d, and 292 e. However,it is understood that more or fewer washes are within the scope of thisinvention. If more washes are desired, more chambers 292 may beprovided. Alternatively, each chamber 292 may hold a larger volume offluid and activation of the plungers may force only a fraction of thevolume from the chamber upon each activation.

After washing, elution buffer stored in chamber 292 f is moved viachannel 247 to blister 248, and the magnet is retracted. The solution iscycled between blisters 246 and 248 via channel 252, breaking up thepellet of magnetic beads in blister 246 and allowing the capturednucleic acids to dissociate from the beads and come into solution. Themagnet 850 is once again activated, capturing the magnetic beads inblister 246, and the eluted nucleic acid solution is forced into blister248.

Plunger 268 h is depressed and first-stage PCR master mix from chamber292 h is mixed with the nucleic acid sample in blister 248. Optionally,the mixture is mixed by alternative activation of bladders 848 and 864,forcing the mixture between 248 and 264 via channel 253. After severalcycles of mixing, the solution is contained in blister 264, wherefirst-stage multiplex PCR is performed. If desired, prior to mixing, thesample may be retained in blister 246 while the first-stage PCR mastermix is pre-heated, illustratively by moving the first-stage PCR mastermix into blister 264 or by providing a heater adjacent blister 248. Asdiscussed above, this pre-heating may provide the benefits of hot startPCR. The instrument 800 illustrated in FIG. 8 features Peltier-basedthermal cyclers 886 and 888 which heat and cool the sample. However, itis understood that other heater/cooler devices may be used, as discussedabove. Optionally, mixing between blisters 248 and 264 may continueduring temperature cycling, with thermal cycler 886 positioned to heatand cool both blisters 248 and 264. It has been found that such mixingimproves the first-stage PCR reaction in some embodiments. Also, thermalcycling can be accomplished by varying the temperatures in two or moredifferent blisters, allowing minimal energy expenditure and maximizingthermal cycling speed. For example the temperature can be maintained at95° C. in blister 248, and 65° C. blister 264, and moving the samplebetween these blisters effectively transfers heat into and out of thesample, allowing rapid and accurate thermal cycling. Temperature cyclingis illustratively performed for 15-20 cycles, although other levels ofamplification may be desirable, depending on the application, asdiscussed above. As will be seen below, the second-stage amplificationzone 280 is configured to detect amplification in 18 second-stagereactions. Accordingly, 18 different primer-pairs may be included in thePCR reaction in blister 264.

In an alternative hot start method, pouch 210 is manufactured with theprimers provided in one of the blisters, illustratively blister 264. Inone embodiment, the primers are freeze dried separately and thenintroduced during manufacture into blister 264 as a friable pellet.Prior to first-stage PCR, illustratively the sample is eluted fromblister 246 and pushed to blister 264 to rehydrate the primer pellet.Peltier 886, which is positioned adjacent blisters 248 and 264 is heatedto 48° C., and PCR master mix is pushed to blister 248. After a hold,illustratively for 10 seconds, during which the two blisters reach 48°C., mixing between blisters 248 and 264 begins. Thus, the enzymes anddNTPs remain in blister 248 and most of the sample and the primersremain in blister 264 until the components separately have reached 48°C. It is understood, however, that the choice of 48° C. was made for usewith concurrent first-stage amplification and RT using AMV, which isactive up to 50° C. If RT is not needed or a more thermostable RT enzymeis used, then one or both of the two blisters 248 and 264 may be heatedup to 58° C., or even higher, depending on the primer meltingtemperature or other factors in a particular first-stage amplificationprotocol. It is understood that this hot start method may be used withany embodiment of the present invention.

In an alternative embodiment, to reduce the complexity of thefirst-stage PCR reaction, blister 248 may be divided into two or moreblisters. It is believed that the number of nonspecific products of amultiplex reaction goes up as the square (or possibly higher power) ofthe number of primers in the mixture, while the loss of sensitivity ofan assay is a linear function of the amount of input sample. Thus, forexample, splitting the first stage PCR into two reactions, each of halfthe volume of the single reaction of this embodiment, would reducesensitivity by two-fold but the quantity and complexity of thenonspecific reactions would be ¼ as much. If blister 248 is divided intoor more blisters, blister 264 may be divided into a number of blistersequal to the number of blisters 248. Each respective blister 248 wouldbe connected to its respective blister 264 via a respective channel 253.Each blister 264 would be provided with a pellet comprising a subset ofall primers. Sample from blister 246 would be divided across eachblister 248, each blister 248 would be sealed from all others, andthermal cycling would proceed with each pair of blisters 248 and 264, asdescribed above. After thermal cycling, the sample would be recombinedinto blister 266 or individually sent to separate sets of second-stageblisters.

After first-stage PCR has proceeded for the desired number of cycles,the sample may be diluted as discussed above with respect to theembodiment of FIG. 5, by forcing most of the sample back into blister248, leaving only a small amount, and adding second-stage PCR master mixfrom chamber 292 i. Alternatively, a dilution buffer from 292 i may bemoved to blister 266 via channel 249 and then mixed with the amplifiedsample in blister 264 by moving the fluids back and forth betweenblisters 264 and 266. After mixing, a portion of the diluted sampleremaining in blister 264 is forced away to three-lobed blister 222, nowthe waste receptacle. If desired, dilution may be repeated severaltimes, using dilution buffer from chambers 292 j and 292 k, and thenadding second-stage PCR master mix from chamber 292 g to some or all ofthe diluted amplified sample. It is understood that the level ofdilution may be adjusted by altering the number of dilution steps or byaltering the percentage of the sample discarded prior to mixing with thedilution buffer or second-stage PCR master mix.

If desired, this mixture of the sample and second-stage PCR master mixmay be pre-heated in blister 264 prior to movement to second-stageblisters 282 for second-stage amplification. As discussed above, suchpreheating may obviate the need for a hot-start component (antibody,chemical, or otherwise) in the second-stage PCR mixture.

The illustrative second-stage PCR master mix is incomplete, lackingprimer pairs, and each of the 18 second-stage blisters 282 is pre-loadedwith a specific PCR primer pair. If desired, second-stage PCR master mixmay lack other reaction components, and these components may then bepre-loaded in the second-stage blisters 282 as well. As discussed abovewith the prior examples, each primer pair may be identical to afirst-stage PCR primer pair or may be nested within the first-stageprimer pair. Movement of the sample from blister 264 to the second-stageblisters completes the PCR reaction mixture. Control samples fromchamber 2921 are also moved to control blisters 283 via channel 267. Thecontrol samples may be positive or negative controls, as desired.Illustratively, each pouch would contain control reactions that validatethe operation of each step in the process and demonstrate that positiveresults are not the result of self-contamination with previouslyamplified nucleic acids. However, this is not practical in manyprotocols, particularly for a highly multiplexed reaction. Oneillustrative way of providing suitable controls involves spiking sampleswith a species such as baker's yeast. The nucleic acids are extractedfrom the yeast, alongside other nucleic acids. First- and second-stagePCR reactions amplify DNA and/or RNA targets from the yeast genome.Illustratively, an mRNA sequence derived from a spliced pre-mRNA can beused to generate an RNA-specific target sequence by arranging the primersequences to span an intron. A quantative analysis of the yeast copynumber against reference standards allows substantial validation thateach component of the system is working. Negative control reactions foreach of the many second-stage assays are more problematic. It may bedesirable to run control reactions either in parallel or in a separaterun.

Activation of bladder 882 of bladder assembly 810 seals the samples intotheir respective second-stage blisters 282, 283, and activation ofbladder 880 provides gentle pressure on second-stage blisters 282, 283,to force second-stage blisters 282, 283 into contact with aheater/cooler device. A window 847 positioned over the second-stageamplification zone 280 allows fluorescence monitoring of the arrayduring PCR and during a DNA melting-curve analysis of the reactionproducts.

It is noted that the pouch 210 of FIG. 6 has several unsealed areas,such as unsealed area 255 and unsealed area 256. These unsealed areasform blisters that are not involved in any of the reactions in thisillustrative embodiment. Rather, these unsealed areas are provided inspace between the working blisters and channels. In some manufacturingprocesses, as compared to pouches that are sealed in all unused space,it has been found that fewer leaks sometimes result when unsealed areassuch as 255 and 256 are provided, presumably by reducing problematicwrinkles in the film material. Such unsealed areas optionally may beprovided on any pouch embodiment.

FIG. 8 shows an illustrative apparatus 800 that could be used with pouch210. Instrument 800 includes a support member 802 that could form a wallof a casing or be mounted within a casing. Instrument 800 also includesa second support member 804 that is optionally movable with respect tosupport member 802, to allow insertion and withdrawal of pouch 210.Movable support member 804 may be mounted on a track or may be movedrelative to support member 802 in any of a variety of ways.Illustratively, a lid 805 fits over pouch 210 once pouch 210 has beeninserted into instrument 800. In another embodiment, both supportmembers 802 and 804 may be fixed, with pouch 210 held into place byother mechanical means or by pneumatic pressure.

Illustratively, the bladder assembly 810 and pneumatic valve assembly808 are mounted on movable member 802, while the heaters 886 and 888 aremounted on support member 802. However, it is understood that thisarrangement is illustrative only and that other arrangements arepossible. As bladder assembly 810 and pneumatic valve assembly 808 aremounted on movable support member 804, these pneumatic actuators may bemoved toward pouch 210, such that the pneumatic actuators are placed incontact with pouch 210. When pouch 210 is inserted into instrument 800and movable support member 804 is moved toward support member 802, thevarious blisters of pouch 210 are in a position adjacent to the variouspneumatic bladders of bladder assembly 810 and the various pneumaticpistons of pneumatic valve assembly 808, such that activation of thepneumatic actuators may force liquid from one or more of the blisters ofpouch 210 or may form pinch valves with one or more channels of pouch210. The relationship between the blisters and channels of pouch 210 andthe pneumatic actuators of bladder assembly 810 and pneumatic valveassembly 808 are discussed in more detail below with respect to FIGS. 9and 10.

Each pneumatic actuator has one or more pneumatic fittings. For example,bladder 824 of bladder assembly 810 has pneumatic fitting 824 a andpneumatic piston 843 has its associated pneumatic fitting 843 a. In theillustrative embodiment, each of the pneumatic fittings of bladderassembly 810 extends through a passageway 816 in movable support member804, where a hose 878 connects each pneumatic fitting to compressed airsource 895 via valves 899. In the illustrative embodiment, thepassageways 816 not only provide access to compressed air source 895,but the passageways also aid in aligning the various components ofbladder assembly 810, so that the bladders align properly with theblisters of pouch 210.

Similarly, pneumatic valve assembly 808 is also mounted on movablesupport member 804, although it is understood that other configurationsare possible. In the illustrative embodiment, pins 858 on pneumaticvalve assembly 808 mount in mounting openings 859 on movable supportmember 804, and pneumatic pistons 843, 852, 853, and 862 extend throughpassageways 816 in movable support member 804, to contact pouch 210. Asillustrated, bladder assembly is mounted on a first side 811 of movablesupport member 804 while pneumatic valve assembly 808 is mounted on asecond side 812 of movable support member 804. However, becausepneumatic pistons 843, 852, 853, and 862 extend through passageways 816,the pneumatic pistons of pneumatic valve assembly 808 and the pneumaticbladders of bladder assembly 810 work together to provide the necessarypneumatic actuators for pouch 210.

As discussed above, each of the pneumatic actuators of bladder assembly810 and pneumatic valve assembly 808 has an associated pneumaticfitting. While only several hoses 878 are shown in FIG. 8, it isunderstood that each pneumatic fitting is connected via a hose 878 tothe compressed gas source 895. Compressed gas source 895 may be acompressor, or, alternatively, compressed gas source 895 may be acompressed gas cylinder, such as a carbon dioxide cylinder. Compressedgas cylinders are particularly useful if portability is desired. Othersources of compressed gas are within the scope of this invention.

Several other components of instrument 810 are also connected tocompressed gas source 895. Magnet 850, which is mounted on a first side813 of support member 802, is illustratively deployed and retractedusing gas from compressed gas source 895 via hose 878, although othermethods of moving magnet 850 are known in the art. Magnet 850 sits inrecess 851 in support member 802. It is understood that recess 851 canbe a passageway through support member 802, so that magnet 850 cancontact blister 246 of pouch 210. However, depending on the material ofsupport member 802, it is understood that recess 851 need not extend allthe way through support member 802, as long as when magnet 850 isdeployed, magnet 850 is close enough to provide a sufficient magneticfield at blister 246, and when magnet 850 is retracted, magnet 850 doesnot significantly affect any magnetic beads present in blister 246.While reference is made to retracting magnet 850, it is understood thatan electromagnet may be used and the electromagnet may be activated andinactivated by controlling flow of electricity through theelectromagnet. Thus, while this specification discusses withdrawing orretracting the magnet, it is understood that these terms are broadenough to incorporate other ways of withdrawing the magnetic field. Itis understood that the pneumatic connections may be pneumatic hoses orpneumatic air manifolds, thus reducing the number of hoses or valvesrequired.

The various pneumatic pistons 868 of pneumatic piston array 869, whichis mounted on support 802, are also connected to compressed gas source895 via hoses 878. While only two hoses 878 are shown connectingpneumatic pistons 868 to compressed gas source 895, it is understoodthat each of the pneumatic pistons 868 are connected to compressed gassource 895. Twelve pneumatic pistons 868 are shown. When the pouch 210is inserted into instrument 800, the twelve pneumatic pistons 868 arepositioned to activate their respective twelve plungers 268 of pouch210. When lid 805 is closed over pouch 210, a lip 806 on lid 805provides a support for fitment 290, so that as the pneumatic pistons 868are activated, lid 805 holds fitment 290 in place. It is understood thatother supports for fitment 290 are within the scope of this invention.

A pair of heating/cooling devices, illustratively Peltier heaters, aremounted on a second side 814 of support 802. First-stage heater 886 ispositioned to heat and cool the contents of blister 264 for first-stagePCR. Second-stage heater 888 is positioned to heat and cool the contentsof second-stage blisters 282 and 283 of pouch 210, for second-stage PCR.It is understood, however, that these heaters could also be used forother heating purposes, and that other heaters may be included, asappropriate for the particular application.

If desired, a feedback mechanism (not shown) may be included ininstrument 800 for providing feedback regarding whether the sample hasactually been forced into a particular blister. Illustrative feedbackmechanisms include temperature or pressure sensors or optical detectors,particularly if a fluorescent or colored dye is included. Such feedbackmechanisms illustratively may be mounted on either of support members802 or 804. For example, a pressure sensor may be mounted on support 802adjacent the location of blister 264. When the sample is supposedlymoved to blister 264, if the pressure sensor is depressed, then sampleprocessing is allowed to continue. However, if the pressure sensor isnot depressed, then sample processing may be stopped, or an errormessage may be displayed on screen 892. Any combination or all of theblisters may have feedback mechanisms to provide feedback regardingproper movement of the sample through the pouch.

When fluorescent detection is desired, an optical array 890 may beprovided. As shown in FIG. 8, optical array 890 includes a light source898, illustratively a filtered LED light source, filtered white light,or laser illumination, and a camera 896. A window 847 through movablesupport 804 provides optical array 890 with access to second-stageamplification zone 280 of pouch 210. Camera 896 illustratively has aplurality of photodetectors each corresponding to a second-stage blister282, 823 in pouch 210. Alternatively, camera 896 may take images thatcontain all of the second-stage blisters 282, 283, and the image may bedivided into separate fields corresponding to each of the second-stageblisters 282, 283. Depending on the configuration, optical array 890 maybe stationary, or optical array 890 may be placed on movers attached toone or more motors and moved to obtain signals from each individualsecond-stage blister 282, 283. It is understood that other arrangementsare possible.

As shown, a computer 894 controls valves 899 of compressed air source895, and thus controls all of the pneumatics of instrument 800. Computer894 also controls heaters 886 and 888, and optical array 890. Each ofthese components is connected electrically, illustratively via cables891, although other physical or wireless connections are within thescope of this invention. It is understood that computer 894 may behoused within instrument 890 or may be external to instrument 890.Further, computer 894 may include built-in circuit boards that controlsome or all of the components, and may also include an externalcomputer, such as a desktop or laptop PC, to receive and display datafrom the optical array. An interface 893, illustratively a keyboardinterface, may be provided including keys for inputting information andvariables such as temperatures, cycle times, etc. Illustratively, adisplay 892 is also provided. Display 892 may be an LED, LCD, or othersuch display, for example.

FIG. 9 shows the relationship between bladder assembly 810 and pouch 210during operation of instrument 800. Bladder assembly comprisessub-assemblies 815, 817, 818, 819, and 822. Because bladder 809 ofbladder sub-assembly 815 is large, bladder sub-assembly 815illustratively has two pneumatic fittings 815 a and 815 b. Bladder 809is used to close off chambers 292 (as shown in FIG. 6) from the plasticfilm portion 217 of pouch 210. When one of the plungers 268 isdepressed, one or both of pneumatic fittings 815 a and 815 b permitbladder 809 to deflate. After the fluid from one of the chambers 292passes through, bladder 809 is re-pressurized, sealing off channels 214,236, 245, 247, and 249. While illustrative bladder sub-assembly 815 hasonly one bladder 809, it is understood that other configurations arepossible, illustratively where each of channels 214, 236, 245, 247, and249 has its own associated bladder or pneumatic piston. Bladdersub-assembly 822 illustratively comprises three bladders 824, 826, and828. As discussed above, bladders 824, 824, and 828 drive thethree-lobed blister 222 for cell lysis. As illustrated, bladders 824,826, and 828 are slightly larger than their corresponding blisters 224,226, 228. It has been found that, upon inflation, the surface of thebladders can become somewhat dome-shaped, and using slightly oversizedbladders allows for good contact over the entire surface of thecorresponding blister, enabling more uniform pressure and betterevacuation of the blister. However, in some circumstances, completeevacuation of individual blisters may or may not be desired, and largeror smaller-sized bladders may be used to control the blister volumeevacuated. Bladder sub-assembly 817 has four bladders. Bladder 836functions as a pinch-valve for channel 236, while bladders 844, 848, and866 are configured to provide pressure on blisters 244, 248, and 266,respectively. Bladder sub-assembly 818 has two bladders 846 and 864,which are configured to provide pressure on blisters 246 and 264,respectively. Finally, bladder sub-assembly 819 controls second-stageamplification zone 280. Bladder 865 acts as a pinch valve for channels265 and 267, while bladder 882 provides gentle pressure to second-stageblisters 282 and 283, to force second-stage blisters into close contactwith heater 888. While bladder assembly 810 is provided with fivesub-assemblies, it is understood that this configuration is illustrativeonly and that any number of sub-assemblies could be used or that bladderassembly 810 could be provided as a single integral assembly.

FIG. 10 similarly shows the relationship between pneumatic valveassembly 808 and pouch 210 during operation of instrument 800. Ratherthan bladders, pneumatic valve assembly 808 has four pneumatic pistons842, 852, 853, and 862. These pneumatic pistons 842, 852, 853, and 862,each driven by compressed air, provide directed pressure on channels242, 252, 253, and 262. Because the pistons are fairly narrow indiameter, they can fit between bladder sub-assembly 817 and bladdersub-assembly 818 to provide pinch valves for channels 242, 252, 253, and262, allowing channels 242, 252, 253, and 262 to be fairly short.However, if desired, pneumatic pistons 842, 852, 853, and 862 could bereplaced by bladders, which may be included in bladder assembly 810,obviating the need for pneumatic valve assembly 808. It is understoodthat any combination of bladders and pneumatic pistons are within thescope of this invention. It is also understood that other methods ofproviding pressure on the channels and blisters of pouch 210, as areknown in the art, are within the scope of this invention.

FIG. 12 shows a pouch 510 that is similar to pouch 210 of FIG. 6.Fitment 590, with entry channels 515 a through 5151, is similar tofitment 290, with entry channels 215 a through 2151. Blisters 544, 546,548, 564, and 566, with their respective channels 538, 543, 552, 553,562, and 565 are similar to blisters 244, 246, 248, 264, and 266, withtheir respective channels 238, 243, 252, 253, 262, and 265 of pouch 210.The channels 245, 247, and 249 of pouch 210 have been somewhatreconfigured as channels 545 a-c, 547 a-b, and 548 a-c on pouch 510; therespective channels of 510 are somewhat shorter than their counterpartchannels on pouch 210. However, it is understood that channelconfigurations are illustrative only, and that various channelconfigurations are within the scope of this invention.

There are two main differences between pouch 510 of FIG. 12 and pouch210 of FIG. 6. First, three-lobed blister 222 has been replaced by lysisblister 522. Lysis blister 522 is configured for vortexing via impactionusing rotating blades or paddles 21 attached to electric motor 19, asshown in FIG. 2 b. Since this method of lysing does not rely onalternating pressure of pneumatic pistons, only a single-lobed blisteris shown. Because lysis blister 522 has only a single lobe, bothchannels 514 and 536 lead to the single lobe of lysis blister 522. It isunderstood that lysis blister 522 may be used in any of the pouchembodiments described herein. It is further understood that lysisassembly 810 illustratively may be modified to replace bladders 824,826, and 828 of bladder sub-assembly 822 with a single bladderconfigured for blister 522. Conversely, a three-lobed blister, asdescribed in various other embodiments above, may be used in pouch 510.Lysis blister 522 may be provided with an optional reinforcing patch523, illustratively attached using adhesive or lamination to theexterior surface of lysis blister 522. Reinforcing patch 523 aids inminimizing tearing of pouch 510 due to repeated contact by paddles 21.FIG. 13 shows an electric motor, illustratively a Mabuchi RC-280SA-2865DC Motor (Chiba, Japan), mounted on second support member 804. In oneillustrative embodiment, the motor is turned at 5,000 to 25,000 rpm,more illustratively 10,000 to 20,000 rpm, and still more illustrativelyapproximately 15,000 to 18,000 rpm. For the Mabuchi motor, it has beenfound that 7.2V provides sufficient rpm for lysis. It is understood,however, that the actual speed may be somewhat slower when the blades 21are impacting pouch 510. Other voltages and speeds may be used for lysisdepending on the motor and paddles used. Optionally, controlled smallvolumes of air may be provided into the bladder adjacent lysis blister522. It has been found that in some embodiments, partially filling theadjacent bladder with one or more small volumes of air aids inpositioning and supporting lysis blister during the lysis process.Alternatively, other structure, illustratively a rigid or compliantgasket or other retaining structure around lysis blister 522, can beused to restrain pouch 510 during lysis.

The second main difference between pouch 510 of FIG. 12 and pouch 210 ofFIG. 6 is that the blisters 281, 282, and 283 of second-stageamplification zone 280 have been replaced by high density array 581 insecond-stage amplification zone 580. High density array 581 comprises aplurality of second-stage wells 582, illustratively 50 or more wells,and even more illustratively 120 or more wells. Embodiments with moresecond-stage wells 582, illustratively about 200, about 400, or evenabout 500 or more are within the scope of this invention. Otherconfigurations are within the scope of this invention as well.Additional second-stage wells 582 may be added by making wells 582smaller, by making high density array 581 larger, or a combinationthereof. For second-stage PCR, each of the wells may contain a pair ofprimers. It is understood that one or more wells may be used forpositive or negative controls.

Cross-contamination between wells as the wells are filled with thediluted first-stage amplification product in blister 566 can be a majorproblem. Cross-contamination was controlled in pouch 210 by filling eachsecond-stage blister through a separate branch of channel 265 and thensealing with bladder 882, illustrated in FIG. 9. With high density array581, wherein fluid may fill some or all of blister 584,cross-contamination between wells must also be controlled. In oneembodiment, the second-stage PCR primers may be bound covalently ornon-covalently to the inside surface of each well, thus functioning muchlike a

PCR chip. However, in many embodiments it is desirable to controlcross-contamination between wells without tethering the PCR primers tothe wells. Controlling cross-contamination between wells can bedifficult in an embodiment wherein the fluid from blister 566 is movedto wells 582 by flowing across a first surface 581 a of high densityarray 581. There are several desirable features for successful loadingof the second-stage amplification zone 580. First, it is desirable thatthe incoming fluid from blister 566 fill substantially all of the wells582 to substantially the same level. An unfilled well would produce afalse negative signal. Second, it is desirable that the process offilling the wells 582 should not cause the primers in the well to leakout. Loss of primers from one well can limit the efficiency of the PCRreaction in that well and can contaminate neighboring wells. Third,after the wells 582 have been filled and PCR started, it is desirablethat the wells be completely sealed from each other. Amplicon leakageout of one well and into another well can lower signal in the first welland raise signal in the second well, potentially leading to a falsenegative in the first well and a false positive in the second well.Further, for certain kinds of controls, it is important that amplicongenerated in one well not enter another well where it can be furtheramplified.

Solutions to this problem include use of a barrier layer. In oneexample, the barrier layer is a physical barrier that is provided toallow for rapid loading of the wells and for rapid sealing from the bulkfluid. In another example, combined chemical and physical barriers areused, wherein the physical barrier is used to seal the wells and thenthe chemical barrier conditionally releases the oligonucleotide primersinto the well solution, for example by heating, slow release, orenzymatic digestion. Well depth or channel length to each well also maybe used to control release of the reagents from the wells. Other meansfor loading high density array 581 are possible.

FIG. 14 shows an illustrative embodiment of second-stage 580 using aphysical barrier. Sandwiched between first layer 518 and second layer519 of pouch 510 is high density array 581, with wells 582. Piercedlayer 585, with piercings 586, is provided on one side of high densityarray 581 to act as the physical barrier, and a second layer 587, isprovided on the opposite side of high density array 581 to form thebottom of wells 582. Illustratively, pierced layer 585 and second layer587 are plastic films that have been sealed to high density array 581,illustratively by heat sealing, although it is understood that othermethods of sealing may be employed. It is also understood that thematerial used for high density array 581 and the material used forpierced layer 585 and second layer 587 should be compatible with eachother, with the sealing method, and with the chemistry being used. Whenused for PCR, examples of compatible plastics that can used for highdensity array 581 and can be heat-sealed are PE, PP, Monprene®, andother block copolymer elastomers. If fluorescent dyes are used in thedetection chemistry, it may be desirable for high density array 581 tobe formed from black or other relatively fluorescently opaque materials,to minimize signal bleed from one well 582 to its neighboring wells andfor at least one of layers 585 and 587 to be relatively fluorescentlytransparent. For pierced layer 585 and second layer 587, laminates of astrong engineering plastic such as Mylar® or PET with heat-sealableplastic layers such as PE, PP and Dupont Surlyn® may be used. Foradhesive-based systems, rigid engineering plastics such as PET orpolycarbonate may be used to form high density array 581 and films ofPCR-compatible plastics are then used as pierced layer 585 and secondlayer 587. In one illustrative embodiment, high density array 581 isformed of black PE, a composite polyethylene/PET laminate (or Xerox® PN104702 hot laminating pouch material) is used for pierced layer 585 andsecond layer 587 which are heat sealed to high density array 581, andcomposite polypropylene/PET is used for first and second layers 518, 519of pouch 510.

It is understood that piercings 586 align with wells 582. It is alsounderstood that piercings 586 are small enough that, absent some force,fluid does not readily flow through piercings 586. Illustrativepiercings may be 0.001-0.1 mm, more illustratively 0.005-0.02 mm, andmore illustratively about 0.01 mm. In the illustrative embodiment,second-stage amplification zone 580 is provided under vacuum, such thatwhen fluid is received from blister 566, the vacuum draws fluid throughpiercings 586 into each well 582. Once the wells 582 are filled, a forceis no longer present to force fluid into or out of the wells 582. Abladder adjacent second-stage amplification zone 580 (not shown, butsimilar in position to bladders 880/882) may then be activated to pressfirst layer 518 against high density array 581 and seal fluid into thewells 582. While first layer 518 of pouch 510 is used to seal the wells582, it is understood that an optional sealing layer may be providedbetween pierced layer 585 and first layer 518.

In one illustrative example, second-stage amplification zone 580 may beprepared as follows. High density array 581 may be prepared by firstpunching, molding, or otherwise forming an array of wells 582 in aplastic sheet (illustratively 0.1 to 1 mm thick). The wells may form anyregular or irregular array that is desired, and may have a volumeillustratively of 0.05 μl to 20 μl, and more illustratively of 0.1 μl to4 μl. One of layers 585 or 587 is then laminated to a first surface 581a of high density array 581, illustratively by heat or adhesive. Asshown in FIG. 15, pierced layer 585 is applied to first surface 581 a.Reagents 589, illustratively elements of the chemistry of the array thatare unique, such as PCR primer pairs, are then spotted into the wellseither manually by pipetting, or automatically (illustratively using x/ypositionable spotters such as pin-spotters, dot-matrix printers,small-volume automatic pipettes, or micro-fluidic micro-contactspotters). Illustrative devices for spotting the reagents are discussedbelow in Example 4. After the reagents 589 have been dried in each well582, the second of layers 585 or 587 is applied to the second surface581 b of array 581. Layer 585 is pierced using an array of smalldiameter needles to form piercings 586. Piercings 586 may be formedeither before or after layer 585 has been fixed to array 581. It isunderstood that spotting can be done on either layer 585 before or afterpiercing or on layer 587. Spotting an array with holes pre-pierced hasnot shown to leak substantially and offers the advantage that theneedles used for piercing are not contaminated by touching the spottedreagents. Alternatively, to minimize the possibility of leakage, and toposition the spotted reagents at the most distant location in the array,it may be desirable to spot the reagents 589 onto second layer 587, sealthe array 581 with layer 585, and then pierce layer 585. In anillustrative example, reagents are spotted onto second layer 587 using aGeSiM A060-324 Nano-Plotter 2.1/E (Grosserkmannsdorf, Germany) or aspotter discussed below in Example 4. Using such a spotter, multiplearrays may be spotted simultaneously.

Once spotted and pierced, array 581 is placed inside layers 518 and 519of pouch 510 and sealed in place, illustratively by heat sealing, usingan adhesive, ultrasonically welding, mechanical closure, or other meansof enclosing array 581 inside pouch 510 within blister 584. It isunderstood that blister 584 is fluidly connected to blister 566 viachannel 565, and that liquid can flow from channel 565 into blister 584and over piercings 586. In one illustrative example, when blister 584 isformed, care is taken to allow a path for air to escape. This can beaccomplished by “waffling” the inside surface of first layer 518adjacent to second-stage amplification zone 580 to imprint the filmmaterial with a pattern of slightly raised texture. This allows air andliquid to pass along the surface of pierced layer 585, and better allowsliquid to reach and fill all of wells 582. The pouch 510 is then placedinside a vacuum chamber and evacuated. Illustratively, when the pressurehas reached approximately 0.3 millibars, a pneumatic cylinder inside thevacuum chamber is actuated, driving down a plunger into fitment 590 toseal channel 567, thereby cutting the path from the array inside thesealed pouch, and the vacuum chamber. A plurality of other plungers arealso driven into fitment 590 to seal the various entry channels 515. Thepouch is removed from the vacuum chamber and may be packaged forlong-term storage in a vacuum-bag.

Pouch 510 may be used in a manner similar to pouch 210. Because array581 is packaged in vacuum, when liquid is moved from blister 566 tosecond-stage amplification zone 580, the liquid sample is drawn throughpiercings 586 and into wells 582. Excess liquid is forced away byinflating a pneumatic bladder over the array and thermal cycling isaccomplished as described above, illustratively by heating and cooling aPeltier element pressed against one side of the array.

As mentioned above, pierced layer 585 may be replaced by a variety ofsuitable physical or chemical barriers. In one illustrative embodimentusing a chemical barrier, pierced layer 585 is omitted, and reagents 589are spotted into wells 582 in a buffer that dissolves relatively slowly.Illustratively, reagents 589 that contain polymers such as PEG, Ficollor polysorbate 20 or sugars such as sucrose, trehalose or mannitol inappropriate concentrations will be compatible with the second-stage PCRreaction and may dissolve more slowly than primers spotted solely inwater or Tris/EDTA. The primers spotted in one of these buffers may beair dried into the wells 582, as described above (it is understood thatin such an embodiment, second layer 587 is affixed to high density array581 for spotting). These same polymers may be used in lyophilization ofenzyme reagents (e.g. the enzymes and buffers used in PCR) to form anopen matrix containing the stabilized enzymes. Thus, the primers spottedin these buffers can be lyophilized in place in the wells 582, leadingto slower but potentially more complete rehydration than with airdrying. When pouch 510 is used, the fluid from blister 566 is driveninto the well by vacuum or pressure and starts to dissolve the primermix. By selecting a buffer that dissolves suitably slowly, when thebladder adjacent second-stage amplification zone 580 is actuated, thecontents of each well 582 is sealed therein prior to any substantialcross-contamination.

Another embodiment uses a matrix that does not dissolve untilsecond-stage amplification zone 580 is heated above a predeterminedtemperature. One example of such a matrix is low melt agarose such asGenePure LowMelt Agarose (ISC Bioexpress). In one example, a 1.5%solution of this agarose melts at 65° C. and gels at 24-28° C. Prior tospotting, reagents 589 illustratively may be warmed to 50° C. and mixedwith this agarose that had already been melted and then spotted intowells 582 in a small volume (illustratively 100 to 500 nl). To keep themixture liquid during spotting, this may have to be done in a cabinetheated above the melting temperature of the agarose. Alternatively, itmay be possible to pipette dilute solutions of the agarose withoutmelting. After the agarose/reagent mixture is spotted, the high densityarray 581 is dried. This can be a simple air drying or theprimer-agarose mixture can contain the sugars and polymers listed aboveso that the reagents can be freeze dried. When pouch 510 is used forPCR, second-stage amplification zone 580 may be heated, illustrativelyto 55° C., as the fluid from blister 566 is moved into high densityarray 581. At this temperature, the agarose does not melt so the primersare not released into solution. Once high density array 581 is filled,the corresponding bladder is inflated to seal the wells. When thetemperature rises above 65° C. in the first denaturation step of thefirst PCR cycle, the agarose containing the primers melts, releasing theprimers into the master mix. Illustratively, thermal cycling never goesbelow 60° C. (or other melting temperature for the agarose) so that theagarose does not gel during thermal cycling. Furthermore, in theillustrative instrument 800 of FIG. 8, the repeated temperature cyclingis driven by heater 888, which is located on one side of the pouch. Itis expected that there will often be a temperature gradient across thePCR solution in wells 582, which should facilitate mixing of the primersby convective fluid flow. Wax may also be used in a similar embodiment.

In a further embodiment, the primers may be conditionally bound to thewells 581, with subsequent releasing of the primers into solution afterthe wells 581 have been filled. Depending upon how the primers areattached to the plastic substrate, the primers may be cleaved using heat(illustratively during the first cycle of the PCR reaction), light(illustratively irradiating through window 847), chemicals (e.g.dithiothreitol together with heat will reduce disulfide bonds that maybe used to link primers to the wells), or enzymes (e.g. site specificproteases such at Tissue Plasminogen Activator can be used to cleave theproper peptide linker attaching primers to the substrate).

In yet another embodiment, a DNase may be injected into second-stageamplification zone 580 subsequent to amplification, to minimize furtherany potential risk of contamination.

It is understood that second-stage amplification zone 580 has beendescribed herein for use with PCR. However, other uses for pouch 510 andsecond-stage amplification zone 580 are within the scope of thisinvention. Further, it is understood that second-stage amplificationzone 580 may be used with or without nucleic acid extraction and a firststage PCR amplification zone. Finally, it is understood thatsecond-stage amplification zone 580 may be used with any of the pouchembodiments described herein.

EXAMPLE 1 Nested Multiplex PCR

A set of reactions was run in a pouch 110 of FIG. 5, on an instrumentsimilar to instrument 800 but configured for pouch 110. To show celllysis and effectiveness of the two-stage nucleic acid amplification, 50μL each of a live culture of S. cerevisaie and S. pombe at log phase wasmixed with 100 μL of a nasopharyngeal aspirate sample from a healthydonor to form the sample, then mixed with 200 μL lysis buffer (6Mguanidine-HCl, 15% TritonX 100, 3M sodium acetate. 300 μL of the 400 μLsample in lysis buffer was then injected into chamber 192 a of pouch110.

The pouch 110 was manufactured with 0.25 g ZS beads sealed inthree-lobed blister 122. Second-stage primers, as discussed below, werealso spotted in blisters 181 and 182 during manufacture of pouch 110.The pouch 110 was loaded as follows:

115 a sample and lysis buffer, as described above,

115 b magnetic beads in the lysis buffer,

115 d-e wash buffer (10 mM sodium citrate),

115 g elution buffer (10 mM Tris, 0.1 mM EDTA)

115 h first-stage PCR buffer:

-   -   0.2 mM dNTPs    -   0.3 μM each primer:        -   Sc1: primers configured for amplifying a portion of the YRA1            nuclear protein that binds to RNA and to MEX67p of S.            cerevisaie. The primers are configured to amplify across an            intron such that amplification of cDNA (mRNA            reverse-transcribed via M-MLV) yields a 180 by amplicon.        -   Sc2: primers configured for amplifying a 121 by region of            the cDNA of MRK1 glycogen synthase kinase 3 (GSK-3) homolog            of S. cerevisaie.        -   Sc3: primers configured for amplifying a 213 by region of            the cDNA of RUB1 ubiquitin-like protein of S. cerevisaie.        -   Sp1: primers configured for amplifying a 200 by region of            the cDNA of sucl-cyclin-dependent protein kinase regulatory            subunit of S. pombe.        -   Sp2: primers configured for amplifying a 180 by region of            the cDNA of sec14-cytosolic factor family of S. pombe.    -   PCR buffer with 3 mM MgCl₂ (without BSA)    -   50 units M-MLV    -   4.5 units Taq:Antibody    -   100 units RNAseOut

115 j-k second-stage PCR buffer

-   -   0.2 mM dNTPs    -   1X LC Green® Plus (Idaho Technology)    -   PCR buffer with 2 mM MgCl₂ (with BSA),    -   4.5 units Taq

115 l second-stage PCR buffer with a sample of the first-stageamplicons.

During manufacture, second-stage blisters 181 and 182 were spotted withnested second-stage primers. Each blister was spotted with one primerpair in an amount to result in a final concentration of about 0.3 μMonce rehydrated with the second-stage PCR buffer. The second-stagenested primers are as follows:

Sc1: primers configured for amplifying an 80 by fragment of the Scl cDNAfirst-stage amplicon.

Sc2: primers configured for amplifying a 121 by fragment of the Scl cDNAfirst-stage amplicon.

Sc3: primers configured for amplifying a 93 by portion of the Scl cDNAfirst-stage amplicon.

Sp1: primers configured for amplifying a 99 by portion of the Scl cDNAfirst-stage amplicon.

Sp2: primers configured for amplifying a 96 by portion of the Scl cDNAfirst-stage amplicon.

There is no overlap between the first-stage and second stage primerpairs for any of the targets. Each pair of primers was spotted into onenegative control blister 181 and two second-stage blisters 182, so thateach second-stage amplification would be run in duplicate, eachduplicate with a negative control.

After loading, activation of the plunger associated with entry channel115 a moved the sample to three-lobed blister 122, activation of theplunger associated with entry channel 115 b moved the magnetic beads toreservoir 101, activation of the plungers associated with entry channels115 d-e moved wash buffer to reservoirs 102 and 103, activation of theplunger associated with entry channel 115 g moved elution buffer toreservoir 104, activation of the plunger associated with entry channel115 h moved first-stage PCR buffer to reservoir 105, activation of theplungers associated with entry channels 115 j-k moved second stage PCRbuffer to reservoirs 106 and 107, and activation of the plungerassociated with entry channel 115 l moved the positive control(second-stage PCR buffer with a sample of previously preparedfirst-stage amplicon) to reservoir 108. In this present example, theplungers associated with entry channels 115 a and 115 b were depressedprior to loading the pouch 110 into the instrument. All other plungerswere depressed sequentially in the instrument during the run, and fluidswere moved to reservoirs 102 through 108 as needed.

Once pouch 110 was placed into the instrument, and beating took placefor ten minutes in the presence of ZS beads, as described above. Oncecell lysis was complete, reservoir 101 was compressed and nucleic acidbinding magnetic beads from reservoir 101 were forced into three-lobedblister 122, where the beads were mixed gently and allowed to incubatefor 5 minutes.

The sample-bead mixture was then moved to blister 144, where themagnetic beads were captured via activation of the magnet. Once themagnet was deployed, bladders adjacent blister 144 were pressurized toforce fluids back to three-lobed blister 122. The captured beads werethen washed as described above, using the wash solution from reservoirs102 and 103. Following washing, the beads were once again captured inblister 144 via activation of the magnet, and the elution buffer storedin reservoir 104 is moved to blister 144, where, after a 2 minuteincubation, the nucleic acids eluted from the beads are then moved toblister 161, as discussed above.

In blister 161, the nucleic acid sample is mixed with first-stage PCRmaster mix from reservoir 105. The sample is then held at 40° C. for 10minutes (during which time M-MLV converts mRNA to cDNA), then 94° C. for2 minutes (to inactivate the M-MLV and remove antibody from taq).Thermal cycling is then 20 cycles of 94° C. for 10 second and 65° C. for20 seconds.

Subsequent to first-stage amplification, the sample is dilutedapproximately 100-fold using the second-stage PCR master mix fromreservoir 106. The sample is then moved to blisters 182, which werepreviously spotted with the second-stage primers, as discussed above.Second-stage PCR buffer was moved from reservoir 181 to negative controlblisters 181, and the positive control mixture was moved to blisters 183from reservoir 108. The samples were denatured for 30 seconds at 94° C.,then amplified for 45 cycles of 94° C. for 5 seconds and 69° C. for 20seconds.

As can be seen in FIG. 11, all target amplicons and the positive controlshowed amplification, while none of the negative controls showedamplification. Each sample was run in replicates. The replicates eachshowed similar amplification (data not shown). It is understood that theS. cerevisaie and S. pombe targets are illustrative only and that othertargets are within the scope of this invention.

EXAMPLE 2 High Density PCR

The above example uses pouch 110 of FIG. 5. Pouch 110 has five negativecontrol blisters 181, five positive control blisters 183, and ten lowvolume sample blisters 182. Pouch 210 of FIG. 6 increased the number oflow volume sample blisters 282 to 18. However, high density array 581 ofpouch 510, shown in FIG. 12 can have 120 or more second-stage wells 582.This increase in the number of second-stage reactions enables a wide setof potential diagnostic and human identification applications withoutthe need to increase the size of the pouch and its instrument. Variousexamples are described herein.

In one example, it is known that standard commercial immunofluorescenceassays for the common respiratory viruses can detect seven viruses:Adenovirus, PIV1, PIV2, PIV3, RSV, Influenza A, and Influenza B. A morecomplete panel illustratively would include assays for additional fiveviruses: coronavirus, human metapneumovirus, BOCAvirus, Rhinovirus andnon-HRV Enterovirus. For highly variable viruses such as Adenovirus orHRV, it is desirable to use multiple primers to target all of thebranches of the virus' lineage (illustratively 4 outer and 4 innerprimer sets respectively). For other viruses such as coronavirus, thereare 4 distinct lineages (229E, NL63, OC43, HKU1) that do not vary fromone season to another, but they have diverged sufficiently enough thatseparate primer sets are required. The illustrative complete respiratoryvirus panel would also target the SARS coronavirus, possibly the avianinfluenza HA and N subtypes, and possibly others. Finally, some of therespiratory viruses show such a high rate of sequence variation that itwould be beneficial to create more than one nested PCR assay for eachsuch virus, thereby minimizing the chance of false negative results dueto sequence variation under the primers. When all of the primer setsdescribed herein are included, such a respiratory virus panel could have80 or more specific amplicons in the second-stage amplification. Thehigh density array 581 could easily accommodate such a panel in a singlepouch 510.

A second application of the high density array 581 of pouch 510 would beto determine the identity and the antibiotic resistance spectrum of themulti-drug resistant bacteria isolated from infected patients. Currentmethods require several days to culture the organism and empiricallytest individual drug resistance profiles. During the time it takes toreceive the results, physicians will often administer broad-spectrumantibiotics, which leads to an increase in multi-drug resistantbacteria. PCR primers have been developed to detect the geneticdeterminants of antibiotic resistance (the antibiotic resistance genesthemselves). However because of the large number of variants of some ofthese genes, a large number of amplicons is required for a completedetermination of the resistance profile. Hujer et. al. describe a panelof 62 PCR assays to identify the resistance genes present inAcinetobacter isolates. Again, the high density array 581 could easilyaccommodate such a panel in a single pouch.

A third example of the utility of the high density array is in the fieldof human identification, illustratively for forensic identification ofhuman remains and for paternity testing. Most of the market in humanidentification is dominated by systems that analyze short tandem repeatsequences (STRs). This analysis has generally required separating therepeats by size, using e.g. capillary electrophoresis. The specializedlaboratory equipment used for this purpose has generally not been fieldportable. There is growing interest in using Single NucleotidePolymorphisms (SNPs) for identity testing, as there are a large set oftechniques for identifying SNPs and some of these are amenable to fielduse. Sanchez et al. have published a set of 52 well-characterized SNPsthat collectively give a very low probability of matching twoindividuals by chance (a mean match probability of at least 5.0×10⁻¹⁹).In practice, it may take two amplicons for each SNP to accurately typeeach locus (see, e.g., Zhou et al.). Thus one pouch 510 with 104second-stage wells 582 could completely type an individual at all of the52 SNP loci.

It is understood that there are cost and workflow advantages gained bycombining assays from different diagnostic applications into one pouch.For example the complete respiratory virus panel could be combined withthe bacterial identification panel. These combinations could simplifymanufacturing, since there are fewer types of pouches to assemble. Theycould also simplify the work of the end user, as there are fewerspecific types of pouches that need to be stocked in a clinic, and alsoreducing the chance of using the wrong pouch for a particular clinicalsample. For some applications, these advantages could offset an increasecost of manufacturing the pouch having a greater number of primer pairs.Thus one pouch 510 with 100 or more second-stage wells 582 could be usedto accommodate multiple panels of assays.

EXAMPLE 3 Process Controls

Controls for highly multiplexed assays can be problematic, especially inclinical diagnostic settings where quality must compete with cost pertest. The high-density array 582 of pouch 510 potentially increases thisproblem because of the increased number of diagnostic targets that canbe assayed in a single run. Various types of controls are discussedherein.

Illustrative process controls include mixing an intact organism, forexample an organism containing an RNA target, into the patient samplebefore injecting the sample into the pouch. Such a target could be anintact RNA bacteriophage (MS2 or Qβ) or an intact plant RNA virus(Tobacco Mosaic Virus) or an mRNA present in an intact yeast. Outerprimers specific for the RNA target would be present in the first-stagePCR and a well 582 containing the inner primers would be present in thehigh density array. Detection of amplification product in this well 582confirms that all of the steps of the process are working correctly. Apost-second-stage amplification melt curve could also be used to verifythat the correct specific product was made. The crossing point (“Cp”)determined from an amplification curve could be used to give aquantitative measure of the integrity of the reagents. For example theCp can be compared to that of other pouches from the same lot run at adifferent time. While an intact organism is used, it is understood thatpurified or isolated nucleic acids may be used if it is not important totest for lysis. In other situations, it may be desirable to use thecontrol to test only the later steps of the analysis. For example,spiking a natural or synthetic nucleic acid template into a well in thehigh density array along with the cognate primers could be used to testthe second-stage PCR reaction, and spiking a nucleic acid template intothe first-stage PCR with the appropriate primers in the first-stage PCRamplification mixture and in a well 582 of the second-stageamplification zone will test both the first- and second-stage PCRreactions.

Process controls such at described above do not test the integrity ofthe primers specific to the target amplicons. One example of a positivecontrol that tests the integrity of the specific primers uses a mixtureof nucleic acids, illustratively synthetic RNAs, as stability andvariability often can be better controlled and these sequences cannot bepresent due to environmental contamination, wherein the mixture containsa nucleic acid for each of the primers present in the particular pouch.In a diagnostic setting, this positive control could be used at the endof a run of pouches used to test patient samples. The mixture isinjected into a pouch, illustratively from the same lot as those usedfor the patient samples, and success is defined by all of the targetamplicons providing a positive result. Negative controls can be done inthe same way; at the end of a run of pouches used to test patientsamples, water or buffer could be injected into a pouch and successdefined by all of the target amplicons providing a negative result.

Individual workflow and protocols in a diagnostic lab may be used todetermine the number of patient sample pouches run before the controlpouches described above are run. Regardless of how frequently orinfrequently the control pouches are run, these controls add to the timeand cost of the total system. For this reason, it would be useful tomake the controls internal to the pouch. The structure of the highdensity array 581 allows for the following novel approach to negativecontrols. In this example, a nucleic acid, illustratively a syntheticamplicon, is spiked into one of the wells 582 a of the high densityarray 581. Primers to amplify this sequence are spiked into this well582 a and into two other wells 582 b and 582 c spaced across the array.Illustratively, the amplicon sequence and primers are artificial anddesigned so that none of the primers used will amplify another target bychance.

When a clean, uncontaminated pouch 510 is run in instrument 800, thewell 582 a containing the synthetic target will generate amplicon andtherefore be called positive. The two other wells 582 a, 582 b thatcontain the corresponding primers should not amplify anything in thesample and thus be called negative. Pouch 510 may be treated further,for additional controls. Illustratively, bladder 880/882 holding thehigh density array against heater 888 is then depressurized and thecontents of the wells 582 are mixed. In one illustrative method, thecontents of the wells 582 are mixed as follows: heater 888 is used tocycle the temperature of the high density array above and below theboiling point of the buffer for a short time (for example three cyclesof 85° C. for 10 sec then 105° C. for to 20 sec). Bubbles of steamgenerated in the wells 582 of high density array 581 should force thecontents of wells 582 out into the second-stage amplification zoneblister 580. Optionally, the contents of the second-stage amplificationzone 580 may be mixed with the contents of rest of the pouch 510 byusing the bladders to move liquid from one end of the pouch 510 to theother. The purpose of these steps is to mix the specific contaminationcontrol amplicon, along with any specific target amplicons throughoutthe pouch.

If the user accidentally opens a pouch after it has been run in thisfashion, then both specific target amplicons and the contaminationcontrol amplicon will be released. If trace amounts of these nucleicacids contaminate a later pouch run, the instrument may detect thecontamination event, as the wells 582 b, 582 c that contained only theprimers specific for the synthetic amplicon will score positive.Software in the instrument will alert the user and the results of therun will be flagged as suspect.

In another method to control contamination, at the end of a run, a DNAdegrading chemical or enzyme may be added to destroy substantially allof the DNA products of the first- and second-stage PCR reactions.Illustratively, this can be done in a way similar to the contaminationdetection method described above, by heating the contents of thesecond-stage array to above the local boiling temperature, thus drawingthe amplified sample out of the wells 582 of the array 851, mixing theheated liquid with the diluted contents of the 1 ^(St) stage reaction,adding an aliquot of a DNA degrading substance, illustratively throughentry channel 515 k, either with or without cooling the mixture, andallowing the DNA degrading reaction to incubate until substantially allof the DNA produced in the PCR reaction has been destroyed. This can beaccomplished using DNAases, acids, or oxidants, as are known in the art.

It is understood that any of the contamination controls described hereinmay be used independently or in any combination thereof.

EXAMPLE 4 Array Loading

FIG. 16 shows another embodiment of a high density array. High densityarray 681 is similar in configuration to that of high density array 581.In this illustrative embodiment, high density array 681 is provided with102 second stage wells 682, arranged in a circular pattern. As shown,array 681 is also provided with a layer 687 affixed thereto, layer 687being similar to second layer 587 discussed above, but array 681 has notyet been provided with a pierced layer. While array 681 is theillustrative array for this example, it is understood that array 581 andother arrangements of high density arrays are within the scope of thisinvention.

As discussed above, commercial spotters, such as the GeSiM A060-324Nano-Plotter 2.1/E (Grosserkmannsdorf, Germany), may be used to loadhigh density array 681. Alternatively, high density array 681 may bespotted using x/y positionable spotters such as pin-spotters, dot-matrixprinters, small-volume automatic pipettes, or micro-fluidicmicro-contact spotters.

FIGS. 17-19 show an illustrative spotter 600. Spotter 600 is providedwith a base 601. Attached to base 601 is pump array 620. Adjacent topump array 620 is loading array 630. Illustrative loading array 630 isconfigured to receive a 96-well plate, although other plateconfigurations are within the scope of this disclosure, including384-well plates, 1536-well plates and plates of other configurations. Itis also understood that one or more vials or reservoirs of otherconfigurations may be used for providing the fluid to be spotted. Asbest seen in FIG. 19, illustrative 96-well plate 636 sits on loadingplatform 635, the 96-well plate 636 being much taller than standardmicrotiter plates, to provide larger reservoirs for spotting multiplearrays. However, standard 96-well plates may be used, as shown in FIG.20. Loading platform 635 may be raised or lowered by action of arm 634.

In FIG. 19, plate 636 is shown in the lowered position. In thisposition, the top 633 of plate 636 is not in contact with, noroverlapping with the tips 642 of straws 641, allowing for easy insertionand removal of plate 636. As shown in FIG. 20, loading platform 635 isin the raised position. A plurality of straws 638 extend through support640 and into each well 637 of plate 636. The straws 638 are tubing thatextend from above support 640 to the bottom of each well 637. In theillustrative embodiment, straws 638 are 25 gauge stainless steel.However, any material may be used that is compatible with the fluid tobe spotted in array 681. Preferably, the material for straws 638 isrigid or semi-rigid, so as to extend to the bottom of wells 637, so thatvirtually all fluid in plate 636 may be used. To aid in using as muchfluid as possible, the tip 642 of each straw 638 is cut at an angle, toprevent straw 638 from sealing against well 637. Illustratively, tip 642may be cut at a 30-60°, more illustratively at a 45° angle, but it isunderstood that the choice of angle may depend on the shape of well 637.For example, if well 637 has a flat bottom, an angle significantly lessthan 30° may be useful, while a generally conical well 637 may require agreater angle. Alternatively, tip 642 may be notched or grooved, or maybe provided with any shape that is not congruent with the bottom of well637, in configurations where sealing against well 637 may beproblematic.

Each of straws 638 extend upward through its respective orifice 641 insupport 640. As shown in the illustrative embodiment, the orifices 641are configured to align with wells 637, and each orifice is sized toallow free movement of straw 638 within orifice 641. A weight 639 isaffixed to the top of each respective straw 638. Each weight 639 biasestip 642 against the bottom of well 637. Cylindrical brass weights areused in the embodiment shown. However, it is understood that this isillustrative only, and that other shapes and materials may be used. Itis desired that the weights 639 be sized larger than the orifices 641,so that when plate 636 is removed, weights 639 rest on support 640 andretain each respective straw 638 in position to enter its respectivewell when the next plate is raised into position.

In the illustrative embodiment, each straw 638 extends through itsrespective weight 639 and is connected to a flexible tube 644. However,it is understood that straw 638 and tube 644 may be connected just belowor within weight 639, or straw 638 may change composition from a morerigid material to a more flexible material. Illustratively, tube 644 isan elastomeric material, for example silicone or polyurethane, with aninner diameter of 0.012 inches and an outer diameter of 0.025 inches.However, it is understood that a variety of materials in other sizes maybe used, depending on the specific application. Illustratively, flexibletube 644 is an elastomeric material, but other materials that aresufficiently flexible without cracking or breaking are within the scopeof this disclosure.

As best seen in FIG. 17, the plurality of tubes 644 extend through pumparray 620 in a parallel arrangement, and extend to print head 660. FIG.21 shows cross-sectional view of pump array 620, wherein first pneumaticseal 623 is located closer to loading array 630, and second pneumaticseal 624 is located closer to print head 660. Tube 644 is sandwichedbetween top support 621 and bottom support 622. Bottom support isprovided with three pneumatic elements. First pneumatic seal 623 andsecond pneumatic seal 624 operate as pinch valves on tube 644, to openand close tube 644 as needed, to allow fluid to flow through tube 644from wells 637 to print head 660. Peristaltic pump 626 is provided witha pump head 625 that is somewhat offset from the mechanical center, asindicated by dashed line 631, thereby forcing fluid in the direction ofprint head, particularly when seal 623 is closed and seal 624 is open.Optionally, pump 626 may be provided on an angle (not shown), such thatpump head 625 first makes contact with tube 644 closer to seal 623,thereby forcing fluid toward seal 624. Openings 627, 628, and 629connect to tubing 612, 613, 614, best seen in FIG. 17, which are thenconnected to a compressed gas source 616. Compressed gas source 616 maybe a compressor, or, alternatively, compressed gas source 616 may be acompressed gas cylinder. Springs (not shown) may be used to bias theseals and pump head in the opposite direction from the direction movedby the compressed gas source, thereby allowing the tubing to recover itsoriginal shape with less resistance. Approximately 50 psi or less isrequired to modulate the appropriate amount of fluid in the illustrativeembodiment. In the illustrative embodiment, fluid in all tubes 644 arepumped simultaneously by a single peristaltic pump 626, thereby movinggenerally uniform amounts of fluid through each tube 644. In theillustrative embodiment, tubes 644 are aligned in a parallelarrangement, and seals 623 and 624 and pump 626 are provided linearly.However, other arrangements are within the scope of this disclosure.

It is understood that the amount of fluid moving through tube 644 toprint head 660 will be determined by the diameter of tube 644, the widthof pump head 625, and the amount of pressure exerted on tube 644 by pump626. Adjusting these parameters is within the scope of this disclosure.Also, it is within the scope of this disclosure to pump fluids throughtube 644 by other means, as are known in the art, for example, rollers,hydraulic pumps, electomechanical pumps, and other metering devices.

Print head 660 is provided with 102 positions 662 that align with the102 wells 682 in high density array 681. Each tube 644 connects to oneof the positions 662. 96-well plate 636 is a rectangular array of 96wells 637, while high density array 681 is a generally circular array of102 wells 682. By using flexible tubing for tubes 644, one can arrangefor transfer of fluid from any of the wells 637 in plate 636 to any ofthe wells 682 in high density array 681, merely by affixing a specifictube 644 to a specific location in print head 660. As shown in FIGS.17-18, there are 96 tubes 644, leaving six positions 662 on print head660 empty. When the fluid is transferred to array 681, six wells 682will remain empty, as seen as the six unfilled wells 682 in FIG. 18.However, it is understood that several straws 638 may be attached to asingle weight 639, such that one well 637 in plate 636 is feeding morethan one location on print head 660. If reactions are to be run induplicate or triplicate in array 681, it may be desirable to place twoor three straws 638 in each well 637 of plate 636, but use fewer wells637 in plate 636. It is understood that a plate with 102 wells may beused to transfer fluid through straws 638. Reconfiguration is within thescope of this disclosure, and reconfiguration may be desirable, ifspotter 600 is used for multiple different arrays 681. Also, it isunderstood that the configuration of print head 660 and correspondingarray 681 is illustrative only, and other configurations of the printhead and array are within the scope of this disclosure, provided thatthe print head generally aligns with a portion or all of the array. Forexample, a print head aligning with array 581, as shown in FIG. 15,would be within the scope of this disclosure. Also, for very highdensity arrays with very small wells, it may not be practical to formdrops small enough not to touch while on the print head. In such cases,it may be desirable to use two or more print heads, and use each to loada respective portion of the array.

As best seen in FIG. 19, each tube 644 extends through print head 660,such that each tube terminates in an orifice 645 just below print heat660. In the illustrative embodiment, a small piece of metal tubing isprovided at the end of each tube 644, which may or may not extendthrough print head 660, to provide this orifice 645, illustratively thesame metal tubing as is used for straws 638. Illustratively, an end 643of orifice 645 is polished to a flat, smooth surface, thus reflectinglight and aiding in visualization of drops 692, as discussed below. Inthe illustrative embodiment, a 250 micron orifice is used, which issized to provide enough surface tension to form and support a drop offluid of 0.1 to 10.0 μl, and more illustratively of 0.5 to 1.0 μl.However, this is exemplary only and other sizes of orifice and drop arewithin the scope of this disclosure. In an alternate embodiment shown inFIG. 27, each tube 644 a may terminate at or within print head 660 a andprint head 660 a may be provided with protrusions 677 a that areillustratively formed integrally with the print head body Protrusions677 a may be painted black or other color or may be provided with ametallic coating, to aid in drop visualization. Orifices 645 a may bemade from or coated with a material conducive for transferring fluids.For example, if the fluid is aqueous, a print head made of hydrophobicmaterials may be used. In one illustrative embodiment, the print head660 a may be made from Teflon® or may be made from another material,such as stainless steel, with a Teflon® coating. Such materials may beused in an embodiment wherein the orifices are provided on the printhead itself (FIG. 27) or in an embodiment wherein each tube 644 extendsthrough print head 660 (FIG. 19), to reduce transfer of the fluids toprint head 660. Further, as shown in FIG. 25, a deionizer 680 may beprovided to reduce static effects and aid in transfer of the drops fromprint head 660.

Upon operation of pump 626, fluid is moved from wells 637 of plate 636,such that drops 692 are formed at each orifice that is connected viatube 644 to its well 637. As best seen in FIG. 17, an imaging system,illustratively comprising a light source and camera, is provided. Thelight source 672, illustratively high incident lights although otherconfigurations are possible, are provided to illuminate drops 692. Acamera 670, illustratively mounted just below opening 668 in base 601,images drops 692. FIG. 22 is a screen shot provided on display 695 (asshown in FIG. 26) of a bottom surface 661 of print head 660, showingdrops 692 attached to each of the 96 orifices that are attached to tubes644. Because there are 102 positions 662 on print head 660, and theillustrative example only uses one straw 638 per well 637, six of thepositions 662 are without drops 692, showing six tube orifices 645. Theremaining 96 orifices are obscured by their respective drops 692. FIG.23 is similar to FIG. 22, except that twenty drops are missing, asindicated by empty positions 691, revealing an extra twenty tubeorifices.

Camera 670 is connected to a processor 694 (see FIG. 26) that isprogrammed to determine whether all drops 692 are sufficient,illustratively by approximating volume (4/3 πr³) and comparing to anacceptable volume range. The programming may also include detection ofbubbles, which may render a drop insufficient. In one illustrativeembodiment, the processor may be programmed with the following steps:

(1) Signals the camera 670 to acquire an image of the print head priorto drop formation,

(2) Qualifies the image to ensure that no drops are present on the printhead 660,

(3) If droplets are present, then the software notifies the user,otherwise it signals the spotter to create drops 692,

(4) Signals the camera 670 to acquire a second image of the print headwith drops 692,

(5) Qualifies the image to ensure proper location and size of the drops692,

(6) If drops 692 fail the qualification in Step (5), then the softwarenotifies the user, otherwise it signals the spotter 600 to print thearray 681,

(7) After the array 681 is printed, the software signals the camera 670to acquire a third image of the print head 660 without drops,

(8) Qualifies the image to ensure that no drops are present on the printhead 660.

(9) The software notifies the user of the status of the qualificationper Step (8).

The image analysis used to qualify the drop presence and absence inSteps 2, 5 and 8, rely on standard binary threshold techniques.

As part of the qualification of the drop formation in Step 5, in oneillustrative embodiment, the image is analyzed to determine the locationand diameter (in pixels) of each drop. The software qualifies that thelocations of the centers of each drop are within a prescribed distanceof the centers of the orifices 641. Next, the software determines theradius of each of the drops and qualifies these as being greater than alower threshold, to ensure that the amount of primer present in eachwell is sufficient for PCR amplification, and less than an upperthreshold, to ensure that the droplet is not deposited outside of thetarget well 682 on the array 681. The lower and upper thresholds may be1%, 2%, 5%, 10%, 15%, 20% or any other percentage to provide a range offluid from the drops that would be tolerable in the application.

As an alternative qualification of the drop formation in Step 5, thedrops may be masked when imaged, each drop with a circular mask. Ratherthan using the radius as a standard, the mask is used. If the dropcompletely fills the circle and does not exceed the circle, then thedrop passes the qualification. If the drop does not completely fill thecircle or exceeds the circle size, then the drop will fail thequalification. The width of the circle line is chosen to provide thethreshold, with a thicker circle line providing a larger range and athinner circle line providing a smaller range.

Alternatively, visual inspection of the image may be used to determinewhether all drops 692 are sufficient. If all drops 692 are sufficient,they may be transferred to array 681. It may be desirable to include acolored or fluorescent dye in the fluid to aid with visualization.Alternatively or in addition, end 643 of orifice 641 may be polishedsmooth to reflect light and aid in visualization. If fluorescent dyesare used for PCR detection, it may be desirable to visualize that dye,or to use another dye for visualization that will not interfere with thefluorescence of PCR. For example, LCGreen® Plus could be used for PCRdetection, while an IRDye® (Licor, Lincoln, Nebr.) could be used fordrop visualization. Other dyes are known in the art. Optionally, printhead 660, array 681, or both may be visualized after transfer todetermine whether sufficient volume from each drop 692 has beentransferred from print head 660.

If the drops 692 are insufficient by failing to meet the predeterminedstandard, the drops may be transferred to array 681, as discussed above,but array 681 may be discarded. Alternatively, insufficient drops 692may be removed from print head 660 by blotting or other means.

To better see other components, placement arm 652 was omitted from FIG.17 and the near side of FIG. 19. However, placement arm is best seen inFIGS. 18, 24, and 25. Placement arm 652 is provided with a platform 653for receiving array 681. Array 681 is provided with one or more,preferably two or more openings 655, through which alignment pins 654extend. The openings 655 may be configured to snap onto alignment pins654 for more secure alignment. Alternatively, a recess may be providedto receive array 681. It is understood that other methods for properplacement of array 681 onto platform 653 are within the scope of thisdisclosure. If the drops are determined to be sufficient by meeting apredetermined standard, placement arm 652 is moved upward, so that eachwell 682 of array 681 is placed in contact with its respective drop 692.Once contact is made, surface tension between each drop 692 and itsrespective orifice 645 is released, and the drop 692 is transferred toits respective well 682. Placement arm 652 may then be lowered, array681 removed, and the process repeated, as long as there is sufficientfluid in each of the wells 637 of plate 636. It is understood that,after drops 692 are transferred, array 681 may be processed as discussedabove with respect to array 581, to become the second-stage for any ofthe pouches described herein.

FIGS. 24-25 show detail of one illustrative placement arm 652. In thisexample, placement arm 652 includes a planar 4-bar mechanism 664,wherein coupler link 656 connects platform 653 to rotating arm 659.Coupler link 656 is attached to follower link 658 at attachment point649, and to rotating arm 659 at attachment point 648. Rotating arm 659is attached to top structure 605 at attachment point 647, and followerlink 658 is attached to top structure 605 at attachment point 650. Theshort link 669 is determined by the distance between attachment points647 and 648. Top structure 605 serves as the ground link of the 4-barmechanism. In the illustrative embodiment, the lengths of links arechosen such that high density array 681 follows a coupler curve thatmoves vertically immediately prior to contacting print head 660. Thisvertical movement is desirable so that each drop 692 makes contact withits respective well 682, while minimizing cross-contamination that mayoccur if the movement immediately prior to contact is not vertical. Thiscoupler curve movement is also useful because the opening 668 for camera670 may be placed directly below print head 660. Platen 663 is joined tocoupler link 656 at rotating joint 646. Rotating joint 646 may beadjusted to provide planar alignment between array 681 and print head660. After alignment is set, rotating joint 646 may be locked down toprovide continued alignment. As shown in FIG. 19, two identical planar4-bar mechanisms 664 are provided, one on either side of top structure605 and are locked in synchrony that rotates the two driver links 657together to provide proper alignment of array 681 at print head 660, asshown in FIG. 25. It is understood that planar 4-bar mechanism 664 isillustrative only, and that other methods of placing an array to printhead 660 are within the scope of this disclosure, including other manualand automatic means.

Print head 660 is affixed to pump plate 667, which is affixed to pumparray 620. Pump plate 667 may be movable relative to pump array 620,allowing for alignment with camera 670. Alternatively, pump plate 667may be provided with multiple attachment locations on pump array 620,allowing for lateral positioning. Such lateral positioning optionallyallows multiple arrays 681, which may be seated adjacent to one anotheron platen 663, to be filled by a single pump head 660, or allows pumphead 660 to print a larger array by first printing in one position andthen printing in a second position.

FIGS. 24-25 show an illustrative arrangement for camera 670. In thisillustrative embodiment, camera 670 is mounted horizontally, and amirror 674 is used to reflect the image received through opening 668.However, it is understood that other arrangements are within the scopeof this invention, including but not limited to omission of the mirrorand vertical mounting of camera 670 below opening 668. A bottom casing604 is provided to protect camera 670. A top casing (not shown) may beprovided to protect many components that are above base 601. However, itis preferable that such top casing allows relatively easy insertion andremoval of array 681 and plate 636.

Returning to FIGS. 22-23 are a plurality of standoffs 675 that are buildinto print head 660, illustratively six standoffs 675, although othernumbers of standoffs may be used. It has been found that when layer 687is affixed to array 681, array 681 often bows somewhat. The standoffs675 function to push array 681 flat against platen 663 during transferof drops 692 to wells 682.

FIG. 26 is a block diagram of a system 698 for spotting arrays. Thesystem 698 includes a computing device 696, which may comprise one ormore processors 694, memories (not shown), computer-readable media (notshown), one or more HMI devices (e.g., input-output devices (not shown),displays 695, printers (not shown), and the like), one or morecommunications interfaces (not shown), and the like. The computingdevice 696 may be communicatively coupled to a spotter 600, which may becoupled to a compressed gas source 616. The computing device may controlone or more components of the system, including the pump array 620, theplacement arm 652, the loading platform 635, the imaging system 676, orany combination. It is understood that the system may be configured suchthat various of the components may be operated manually.

Spotter 600 may be cleaned by insertion of a plate 636 in which allwells 637 contain cleaning solution, and the pump 626 activated untilall cleaning solution has exited the orifices 641. A receptacle may beplaced under print head 660 to collect the cleaning solution.

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Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

1. A spotter device for spotting a plurality of fluids into an array comprising: a plurality of reservoirs provided in a first configuration, each reservoir holding its respective fluid, a print head having a plurality of positions provided in a second configuration, the second configuration being different from the first configuration, a plurality of tubes, each tube configured to provide fluid communication from a reservoir at a first end of the tube to a position in the print head at the second end of the tube, and a pump for pumping fluid through the tubes from the reservoir to the print head.
 2. The spotter device of claim 1, further comprising a support provided above the reservoirs, a plurality of straws, each straw connected to a first end of its respective tube, each straw extending from the first end of its respective tube and through the support and into a reservoir, each straw having a weight positioned above the support to bias the straw to a bottom of the straw's respective reservoir.
 3. The spotter device of claim 2, wherein upon removal of the reservoirs, the weights rest on the support.
 4. The spotter device of claim 2, wherein the straws are rigid or semi-rigid, and the tubes are elastomeric.
 5. The spotter device of claim 1, wherein the pump further comprises: a pump configured for simultaneous pumping of all tubes.
 6. The spotter device of claim 5, wherein the pump is a peristaltic pump provided with an offset pump head.
 7. The spotter device of claim 5, wherein the second end of each tube comprises an orifice, and activation of the pump moves fluid from the reservoirs resulting in a drop of fluid at each orifice.
 8. The spotter device of claim 7, wherein the array is provided with a plurality of wells provided in the second configuration.
 9. The spotter device of claim 8, further comprising a placement arm for receiving the array and moving the array to the print head, such that each drop is transferred to its respective array well.
 10. The spotter device of claim 7, further comprising a camera positioned in visual contact with the print head, the camera further operable to provide an image of the drops.
 11. The spotter device of claim 10, further comprising a CPU, the CPU having software configured to analyze the image, the software operable to determine whether each drop meets a predetermined standard.
 12. The spotter device of claim 10, further comprising a light source to illuminate the drops.
 13. The spotter device of claim 12, wherein the light source is a high-incident light source.
 14. The spotter device of claim 12, wherein each orifice has an end polished to a flat, smooth surface, the end configured for reflecting light from the light source back into its respective drop.
 15. The spotter device of claim 10, wherein the fluid in each of the reservoirs contains a fluorescent dye to aid in imaging the drops.
 16. The spotter device of claim 1, wherein the tubes are removably connected to the print head so that any preselected reservoir in fluid communication with a tube may be set to provide fluid from the preselected reservoir to any position on the print head.
 17. The spotter device of claim 1, wherein the plurality of reservoirs are wells in a 96-well plate.
 18. A method for printing an array having a plurality of wells in a configuration, comprising simultaneously pumping fluid from a plurality of reservoirs to a plurality of positions on a print head to form a plurality of drops on the print head having the same configuration as the array, moving the array into contact with the drops, and simultaneously transferring each respective drop into its respective well.
 19. The method of claim 18, further comprising imaging the drops and determining whether all of the plurality of drops are sufficient prior to moving the array into contact with the drops.
 20. The method of claim 19, wherein if one or more drops are determined not to be sufficient, the array is discarded.
 21. The method of claim 19, wherein if one or more of the drops are determined not to be sufficient, a blotting material is moved into contact with the drops, and the pumping and imaging steps are repeated.
 22. An array produced by the method of claim
 18. 23. A system for delivering one or more liquids into a preselected array of a plurality of wells comprising: a plurality of tubes, each tube having a first end in fluid communication with a reservoir, and a second end terminating in an orifice; a plurality of the reservoirs, the reservoirs provided in a predetermined configuration relative to one another; a print head operable to movably hold each orifice in a predetermined position such that the position of each orifice corresponds to a well in the preselected array of wells; a plurality of straws, each straw having a hollow opening connecting a first end to a second end, the first end fluidly connected to the first end of a corresponding tube and the second end removably in contact with a bottom portion of a corresponding reservoir; and a metering device operable to urge a preselected amount of fluid from the second end of each straw through its corresponding tube, and out its corresponding orifice.
 24. The system of claim 23, wherein the metering device is located between the first end and the second end of the plurality of tubes.
 25. The system of claim 23, further comprising an array of a plurality of wells, each well operable to receive the preselected amount of liquid from a corresponding orifice.
 26. The system of claim 23, wherein the number of wells exceeds the number of reservoirs.
 27. The system of claim 26, wherein each reservoir comprises a bottom portion operable to hold a preselected amount of liquid, and further comprising a top portion operable to receive one or more straws.
 28. The system of claim 27, further comprising one or more valves in fluid communication with each tube, said one or more valve operable to stop fluid travel through each tube.
 29. The system of claim 23, wherein each orifice comprises an inner lumen and an outer surface, and wherein the orifice is sized and shaped to retain a droplet of a preselected volume of the liquid adjacent to its outer surface until surface tension is released.
 30. The system of claim 29, wherein preselected array of a plurality of wells is removably held upon a movable platform, the movable platform having a first position and a second position relative to the print head; the first position being within close proximity of the print head such that each of the plurality of wells is in contact with the droplet retained by the corresponding orifice; and the second position being distal from the print head such that no contact is made between any well and any droplet retained by the corresponding orifice.
 31. The system of claim 30, wherein the first position does not bring any well in contact with any orifice.
 32. The system of claim 23, wherein the metering device is a peristaltic pump.
 33. The system of claim 23, wherein that the position of two or more orifices correspond to a single well in the preselected array of wells.
 34. The system of claim 23, further comprising an imaging system, and a computing device, wherein the computing device interfaces with the imaging system to accept or reject an array of drops at the print head, either prior to or subsequent to transfer to the array of wells.
 35. The system of claim 34, wherein the imaging system determines a size for each drop and each drop is accepted or rejected by comparing the size to a standard.
 36. The system of claim 35, wherein the size is a radius.
 37. The system of claim 23, further comprising a deionizer configured to aid in transfer of the fluid from print head to the array of wells. 